Novel pro258 gene disruptions, and methods relating thereto

ABSTRACT

The present invention relates to transgenic animals, as well as compositions and methods relating to the characterization of gene function. Specifically, the present invention provides transgenic mice comprising disruptions in PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 genes. Such in vivo studies and characterizations may provide valuable identification and discovery of therapeutics and/or treatments useful in the prevention, amelioration or correction of diseases or dysfunctions associated with gene disruptions such as neurological disorders; cardiovascular, endothelial or angiogenic disorders; eye abnormalities; immunological disorders; oncological disorders; bone metabolic abnormalities or disorders; lipid metabolic disorders; or developmental abnormalities.

FIELD OF THE INVENTION

The present invention relates to compositions, including transgenic andknockout animals and methods of using such compositions for thediagnosis and treatment of diseases or disorders.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesion molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immuno-adhesions, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

Given the importance of secreted and membrane-bound proteins inbiological and disease processes, in vivo studies and characterizationsmay provide valuable identification and discovery of therapeutics and/ortreatments useful in the prevention, amelioration or correction ofdiseases or dysfunctions. In this regard, genetically engineered micehave proven to be invaluable tools for the functional dissection ofbiological processes relevant to human disease, including immunology,cancer, neuro-biology, cardiovascular biology, obesity and many others.Gene knockouts can be viewed as modeling the biological mechanism ofdrug action by presaging the activity of highly specific antagonists invivo. Knockout mice have been shown to model drug activity; phenotypesof mice deficient for specific pharmaceutical target proteins canresemble the human clinical phenotype caused by the correspondingantagonist drug. Gene knockouts enable the discovery of the mechanism ofaction of the target, the predominant physiological role of the target,and mechanism-based side-effects that might result from inhibition ofthe target in mammals. Examples of this type include mice deficient inthe angiotensin converting enzyme (ACE) [Esther, C. R. et al., Lab.Invest., 74:953-965 (1996)] and cyclooxygenase-1 (COX1) genes[Langenbach, R. et al., Cell, 83:483-492 (1995)]. Conversely, knockingthe gene out in the mouse can have an opposite phenotypic effect to thatobserved in humans after administration of an agonist drug to thecorresponding target. Examples include the erythropoietin knockout [Wu,C. S. et al., Cell, 83:59-67 (1996)], in which a consequence of themutation is deficient red blood cell production, and the GABA(A)-R-β3knockout [DeLorey, T. M., J. Neurosci., 18:8505-8514 (1998)], in whichthe mutant mice show hyperactivity and hyper-responsiveness. Both thesephenotypes are opposite to the effects of erythropoietin andbenzodiazepine administration in humans. A striking example of a targetvalidated using mouse genetics is the ACC2 gene. Although the human ACC2gene had been identified several years ago, interest in ACC2 as a targetfor drug development was stimulated only recently after analysis of ACC2function using a knockout mouse. ACC2 mutant mice eat more than theirwild-type littermates, yet burn more fat and store less fat in theiradipocytes, making this enzyme a probable target for chemical antagonismin the treatment of obesity [Abu-Elheiga, L. et al., Science,291:2613-2616 (2001)].

In the instant application, mutated gene disruptions have resulted inphenotypic observations related to various disease conditions ordysfunctions including: CNS/neurological disturbances or disorders suchas anxiety; eye abnormalities and associated diseases; cardiovascular,endothelial or angiogenic disorders including atherosclerosis; abnormalmetabolic disorders including diabetes and dyslipidemias associated withelevated serum triglycerides and cholesterol levels; immunological andinflammatory disorders; oncological disorders; bone metabolicabnormalities or disorders such as arthritis, osteoporosis andosteopetrosis; or a developmental disease such as embryonic lethality.

SUMMARY OF THE INVENTION A. Embodiments

The invention provides an isolated nucleic acid molecule comprising anucleotide sequence that encodes a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide having a full-length amino acidsequence as disclosed herein, an amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneprotein, with or without the signal peptide, as disclosed herein or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide cDNA as disclosed herein, the codingsequence of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide lacking the signal peptide as disclosedherein, the coding sequence of an extracellular domain of atransmembrane PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides are contemplated.

The invention also provides fragments of a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidethat may optionally encode a polypeptide comprising a binding site foran anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody or as antisenseoligonucleotide probes. Such nucleic acid fragments usually are or areat least about 10 nucleotides in length, alternatively are or are atleast about 15 nucleotides in length, alternatively are or are at leastabout 20 nucleotides in length, alternatively are or are at least about30 nucleotides in length, alternatively are or are at least about 40nucleotides in length, alternatively are or are at least about 50nucleotides in length, alternatively are or are at least about 60nucleotides in length, alternatively are or are at least about 70nucleotides in length, alternatively are or are at least about 80nucleotides in length, alternatively are or are at least about 90nucleotides in length, alternatively are or are at least about 100nucleotides in length, alternatively are or are at least about 110nucleotides in length, alternatively are or are at least about 120nucleotides in length, alternatively are or are at least about 130nucleotides in length, alternatively are or are at least about 140nucleotides in length, alternatively are or are at least about 150nucleotides in length, alternatively are or are at least about 160nucleotides in length, alternatively are or are at least about 170nucleotides in length, alternatively are or are at least about 180nucleotides in length, alternatively are or are at least about 190nucleotides in length, alternatively are or are at least about 200nucleotides in length, alternatively are or are at least about 250nucleotides in length, alternatively are or are at least about 300nucleotides in length, alternatively are or are at least about 350nucleotides in length, alternatively are or are at least about 400nucleotides in length, alternatively are or are at least about 450nucleotides in length, alternatively are or are at least about 500nucleotides in length, alternatively are or are at least about 600nucleotides in length, alternatively are or are at least about 700nucleotides in length, alternatively are or are at least about 800nucleotides in length, alternatively are or are at least about 900nucleotides in length and alternatively are or are at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide-encoding nucleotide sequencewith other known nucleotide sequences using any of a number of wellknown sequence alignment programs and determining which PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide-encoding nucleotide sequences arecontemplated herein. Also contemplated are the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide fragmentsencoded by these nucleotide molecule fragments, preferably those PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide fragments that comprise a binding site for an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody.

The invention provides isolated PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides encoded by any of the isolatednucleic acid sequences hereinabove identified.

In a certain aspect, the invention concerns an isolated PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide,comprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidecomprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

In one aspect, the invention concerns PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 variant polypeptides which are orare at least about 10 amino acids in length, alternatively are or are atleast about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600 amino acids in length, or more. Optionally, PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779variant polypeptides will have or have no more than one conservativeamino acid substitution as compared to the native PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesequence, alternatively will have or will have no more than 2, 3, 4, 5,6, 7, 8, 9, or 10 conservative amino acid substitution as compared tothe native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide sequence.

In a specific aspect, the invention provides an isolated PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide and recovering the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide and recovering the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide from the cell culture.

The invention provides agonists and antagonists of a native PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide as defined herein. In particular, the agonist or antagonistis an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody or a smallmolecule.

The invention provides a method of identifying agonists or antagoniststo a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide which comprise contacting the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide with a candidate molecule and monitoring a biologicalactivity mediated by said PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Preferably, the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide is a native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide.

The invention provides a composition of matter comprising a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, or an agonist or antagonist of a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide as hereindescribed, or an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

The invention provides the use of a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, or an agonist orantagonist thereof as hereinbefore described, or an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention provides vectors comprising DNA encoding any of the hereindescribed polypeptides. Host cell comprising any such vector are alsoprovided. By way of example, the host cells may be CHO cells, E. coli,or yeast. A process for producing any of the herein describedpolypeptides is further provided and comprises culturing host cellsunder conditions suitable for expression of the desired polypeptide andrecovering the desired polypeptide from the cell culture.

The invention provides chimeric molecules comprising any of the hereindescribed polypeptides fused to a heterologous polypeptide or amino acidsequence. Example of such chimeric molecules comprise any of the hereindescribed polypeptides fused to an epitope tag sequence or a Fc regionof an immunoglobulin.

The invention provides an antibody which binds, preferably specifically,to any of the above or below described polypeptides. Optionally, theantibody is a monoclonal antibody, humanized antibody, antibody fragmentor single-chain antibody.

The invention provides oligonucleotide probes which may be useful forisolating genomic and cDNA nucleotide sequences, measuring or detectingexpression of an associated gene or as antisense probes, wherein thoseprobes may be derived from any of the above or below describednucleotide sequences. Preferred probe lengths are described above.

The invention also provides a method of identifying a phenotypeassociated with a disruption of a gene which encodes for a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal; and

(c) comparing the measured physiological characteristic with that of agender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal. In one aspect, the non-human transgenic animal is a mammal. Inanother aspect, the mammal is a rodent. In still another aspect, themammal is a rat or a mouse. In one aspect, the non-human transgenicanimal is heterozygous for the disruption of a gene which encodes for aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide. In another aspect, the phenotype exhibited by thenon-human transgenic animal as compared with gender matched wild-typelittermates is at least one of the following: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In still yet another aspect, the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still another aspect, the immunological disorders are consistent withsystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In still another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety-like response during open fieldactivity testing; abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; increased exploratoryactivity during open-field testing; increased stress inducedhyperthermia; enhanced motor coordination during inverted screentesting; impaired motor coordination during inverted screen testing;increase in retinal artery tortuosity; retinal degeneration marked byattenuated retinal vessels; opthamological abnormalities; increased meansystolic blood pressure; increased mean fasting serum glucose levels;decreased mean serum glucose levels; increased mean serum cholesterollevels; increased mean serum triglyceride levels; decreased mean serumcholesterol levels; decreased mean serum triglyceride levels; enhancedglucose tolerance; impaired glucose tolerance; increased mean seruminsulin levels; decreased mean serum insulin levels; increased uric acidlevels; decreased serum phosphate levels; increased alkaline phosphataselevels and increased alanine amino transferase levels; liver disease;increased mean percentage of CD25+ in both spleen and lymph nodes;decreased mean percentage of natural killer cells; decreased meanpercentage of CD21HiCD23Med cells in spleen and lymph nodes; increasedmean percentage of CD4 cells and decreased mean percentage of B cells;increased mean percentage of CD8+ cells; decreased mean percentage ofeosinophils; decreased mean serum IgG1 response to an ovalbuminchallenge; decreased mean serum IgG2a response to an ovalbuminchallenge; increased mean serum IgG1 response to an ovalbumin challenge;increased mean serum IgG2a response to an ovalbumin challenge; increasedmean serum MCP-1 response to a LPS challenge; increased mean serumTNF-alpha response to a LPS challenge; decreased mean serum MCP-1response to a LPS challenge; decreased mean serum IL-6 response to a LPSchallenge; decreased TNF-alpha response to a LPS challenge; increasedmean serum IL6 response to a LPS challenge; increased mean plateletcounts; decreased mean total white blood cell (WBC) counts; decreasedabsolute lymphocyte counts; decreased absolute monocyte counts;decreased skin fibroblast proliferation; increased skin fibroblastproliferation; increased mean percent of total body fat and total fatmass; increased mean body weight; increased mean body length; increasedorgan weights; increased total tissue mass (TTM); increased lean bodymass (LBM); increased bone mineral density (BMD) in total body, femurand vertebrae; increased bone mineral content (BMC) in total body, femurand vertebrae; increased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; increased mean femoral midshaft corticalthickness and cross-sectional area; increased mean vertebral trabecularbone volume, number and connectivity density; decreased mean percent oftotal body fat and total fat mass; decreased mean body weight; decreasedmean body length; decreased total tissue mass (TTM); decreased lean bodymass (LBM); decreased bone mineral density (BMD) in total body, femurand vertebrae; decreased bone mineral content (BMC) in total body, femurand vertebrae; decreased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; decreased mean femoral midshaft corticalthickness and cross-sectional area; decreased mean vertebral trabecularbone volume, number and connectivity density; severe depletion ofabdominal and subcutaneous body fat deposits; decreased organ weights;growth retardation; hydrocephalus; sebaceous gland hyperplasia andgrowth retardation; apoptosis of olfactory neuroepithelial cells;lymphoid hyperplasia and tissue inflammation; development abnormalities;male infertility; growth retardation with reduced viability; andembryonic lethality.

The invention also provides an isolated cell derived from a non-humantransgenic animal whose genome comprises a disruption of the gene whichencodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. In one aspect, the isolated cell is amurine cell. In yet another aspect, the murine cell is an embryonic stemcell. In still another aspect, the isolated cell is derived from anon-human transgenic animal which exhibits at least one of the followingphenotypes compared with gender matched wild-type littermates: aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality. The invention also provides amethod of identifying an agent that modulates a phenotype associatedwith a disruption of a gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the methodcomprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the test agent modulates the identifiedphenotype associated with gene disruption in the non-human transgenicanimal.

In one aspect, the phenotype associated with the gene disruptioncomprises a neurological disorder; a cardiovascular, endothelial orangiogenic disorder; an eye abnormality; an immunological disorder; anoncological disorder; a bone metabolic abnormality or disorder; a lipidmetabolic disorder; or a developmental abnormality.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In yet another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism, or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In still another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still another aspect, the immunological disorders are consistent withsystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety-like response during open fieldactivity testing; abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; increased exploratoryactivity during open-field testing; increased stress inducedhyperthermia; enhanced motor coordination during inverted screentesting; impaired motor coordination during inverted screen testing;increase in retinal artery tortuosity; retinal degeneration marked byattenuated retinal vessels; opthamological abnormalities; increased meansystolic blood pressure; increased mean fasting serum glucose levels;decreased mean serum glucose levels; increased mean serum cholesterollevels; increased mean serum triglyceride levels; decreased mean serumcholesterol levels; decreased mean serum triglyceride levels; enhancedglucose tolerance; impaired glucose tolerance; increased mean seruminsulin levels; decreased mean serum insulin levels; increased uric acidlevels; decreased serum phosphate levels; increased alkaline phosphataselevels and increased alanine amino transferase levels; liver disease;increased mean percentage of CD25+ in both spleen and lymph nodes;decreased mean percentage of natural killer cells; decreased meanpercentage of CD21HiCD23Med cells in spleen and lymph nodes; increasedmean percentage of CD4 cells and decreased mean percentage of B cells;increased mean percentage of CD8+ cells; decreased mean percentage ofeosinophils; decreased mean serum IgG1 response to an ovalbuminchallenge; decreased mean serum IgG2a response to an ovalbuminchallenge; increased mean serum IgG1 response to an ovalbumin challenge;increased mean serum IgG2a response to an ovalbumin challenge; increasedmean serum MCP-1 response to a LPS challenge; increased mean serumTNF-alpha response to a LPS challenge; decreased mean serum MCP-1response to a LPS challenge; decreased mean serum IL-6 response to a LPSchallenge; decreased TNF-alpha response to a LPS challenge; increasedmean serum IL6 response to a LPS challenge; increased mean plateletcounts; decreased mean total white blood cell (WBC) counts; decreasedabsolute lymphocyte counts; decreased absolute monocyte counts;decreased skin fibroblast proliferation; increased skin fibroblastproliferation; increased mean percent of total body fat and total fatmass; increased mean body weight; increased mean body length; increasedorgan weights; increased total tissue mass (TTM); increased lean bodymass (LBM); increased bone mineral density (BMD) in total body, femurand vertebrae; increased bone mineral content (BMC) in total body, femurand vertebrae; increased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; increased mean femoral midshaft corticalthickness and cross-sectional area; increased mean vertebral trabecularbone volume, number and connectivity density; decreased mean percent oftotal body fat and total fat mass; decreased mean body weight; decreasedmean body length; decreased total tissue mass (TTM); decreased lean bodymass (LBM); decreased bone mineral density (BMD) in total body, femurand vertebrae; decreased bone mineral content (BMC) in total body, femurand vertebrae; decreased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; decreased mean femoral midshaft corticalthickness and cross-sectional area; decreased mean vertebral trabecularbone volume, number and connectivity density; severe depletion ofabdominal and subcutaneous body fat deposits; decreased organ weights;growth retardation; hydrocephalus; sebaceous gland hyperplasia andgrowth retardation; apoptosis of olfactory neuroepithelial cells;lymphoid hyperplasia and tissue inflammation; development abnormalities;male infertility; growth retardation with reduced viability; andembryonic lethality. The invention also provides an agent whichmodulates the phenotype associated with gene disruption. In one aspect,the agent is an agonist or antagonist of a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yetanother aspect, the agonist agent is an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody. In still another aspect, the antagonist agentis an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a method of identifying an agent thatmodulates a physiological characteristic associated with a disruption ofthe gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic exhibited by the non-humantransgenic animal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic exhibited by the non-human transgenic animal that differsfrom the physiological characteristic exhibited by the wild-type animalis identified as a physiological characteristic associated with genedisruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the physiological characteristic associated withgene disruption is modulated.

In one aspect, the non-human transgenic animal exhibits at least one ofthe following physiological characteristics compared with gender matchedwild-type littermates:

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety-like response during open fieldactivity testing; abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; increased exploratoryactivity during open-field testing; increased stress inducedhyperthermia; enhanced motor coordination during inverted screentesting; impaired motor coordination during inverted screen testing;increase in retinal artery tortuosity; retinal degeneration marked byattenuated retinal vessels; opthamological abnormalities; increased meansystolic blood pressure; increased mean fasting serum glucose levels;decreased mean serum glucose levels; increased mean serum cholesterollevels; increased mean serum triglyceride levels; decreased mean serumcholesterol levels; decreased mean serum triglyceride levels; enhancedglucose tolerance; impaired glucose tolerance; increased mean seruminsulin levels; decreased mean serum insulin levels; increased uric acidlevels; decreased serum phosphate levels; increased alkaline phosphataselevels and increased alanine amino transferase levels; liver disease;increased mean percentage of CD25+ in both spleen and lymph nodes;decreased mean percentage of natural killer cells; decreased meanpercentage of CD21HiCD23Med cells in spleen and lymph nodes; increasedmean percentage of CD4 cells and decreased mean percentage of B cells;increased mean percentage of CD8+ cells; decreased mean percentage ofeosinophils; decreased mean serum IgG1 response to an ovalbuminchallenge; decreased mean serum IgG2a response to an ovalbuminchallenge; increased mean serum IgG1 response to an ovalbumin challenge;increased mean serum IgG2a response to an ovalbumin challenge; increasedmean serum MCP-1 response to a LPS challenge; increased mean serumTNF-alpha response to a LPS challenge; decreased mean serum MCP-1response to a LPS challenge; decreased mean serum IL-6 response to a LPSchallenge; decreased TNF-alpha response to a LPS challenge; increasedmean serum IL6 response to a LPS challenge; increased mean plateletcounts; decreased mean total white blood cell (WBC) counts; decreasedabsolute lymphocyte counts; decreased absolute monocyte counts;decreased skin fibroblast proliferation; increased skin fibroblastproliferation; increased mean percent of total body fat and total fatmass; increased mean body weight; increased mean body length; increasedorgan weights; increased total tissue mass (TTM); increased lean bodymass (LBM); increased bone mineral density (BMD) in total body, femurand vertebrae; increased bone mineral content (BMC) in total body, femurand vertebrae; increased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; increased mean femoral midshaft corticalthickness and cross-sectional area; increased mean vertebral trabecularbone volume, number and connectivity density; decreased mean percent oftotal body fat and total fat mass; decreased mean body weight; decreasedmean body length; decreased total tissue mass (TTM); decreased lean bodymass (LBM); decreased bone mineral density (BMD) in total body, femurand vertebrae; decreased bone mineral content (BMC) in total body, femurand vertebrae; decreased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; decreased mean femoral midshaft corticalthickness and cross-sectional area; decreased mean vertebral trabecularbone volume, number and connectivity density; severe depletion ofabdominal and subcutaneous body fat deposits; decreased organ weights;growth retardation; hydrocephalus; sebaceous gland hyperplasia andgrowth retardation; apoptosis of olfactory neuroepithelial cells;lymphoid hyperplasia and tissue inflammation; development abnormalities;male infertility; growth retardation with reduced viability; andembryonic lethality.

The invention also provides an agent that modulates a physiologicalcharacteristic which is associated with gene disruption. In one aspect,the agent is an agonist or antagonist of the phenotype associated with adisruption of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet anotheraspect, the agent is an agonist or antagonist of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. Inyet another aspect, the agonist agent is an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody. In still another aspect, the antagonist agentis an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a method of identifying an agent whichmodulates a behavior associated with a disruption of the gene whichencodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) observing the behavior exhibited by the non-human transgenic animalof (a);

(c) comparing the observed behavior of (b) with that of a gender matchedwild-type animal, wherein the observed behavior exhibited by thenon-human transgenic animal that differs from the observed behaviorexhibited by the wild-type animal is identified as a behavior associatedwith gene disruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the agent modulates the behavior associated withgene disruption.

In one aspect, the observed behavior is an increased anxiety-likeresponse during open field activity testing. In yet another aspect, theobserved behavior is a decreased anxiety-like response during open fieldactivity testing. In yet another aspect, the observed behavior is anabnormal circadian rhythm during home-cage activity testing. In yetanother aspect, the observed behavior is an enhanced motor coordinationduring inverted screen testing. In yet another aspect, the observedbehavior is impaired motor coordination during inverted screen testing.In yet another aspect, the observed behavior includes depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Such disorders include the category defined as “anxiety disorders” whichinclude but are not limited to: mild to moderate anxiety, anxietydisorder due to a general medical condition, anxiety disorder nototherwise specified, generalized anxiety disorder, panic attack, panicdisorder with agoraphobia, panic disorder without agoraphobia,posttraumatic stress disorder, social phobia, social anxiety, autism,specific phobia, substance-induced anxiety disorder, acute alcoholwithdrawal, obsessive compulsive disorder, agoraphobia, monopolardisorders, bipolar disorder I or II, bipolar disorder not otherwisespecified, cyclothymic disorder, depressive disorder, major depressivedisorder, mood disorder, substance-induced mood disorder, enhancement ofcognitive function, loss of cognitive function associated with but notlimited to Alzheimer's disease, stroke, or traumatic injury to thebrain, seizures resulting from disease or injury including but notlimited to epilepsy, learning disorders/disabilities, cerebral palsy. Inaddition, anxiety disorders may apply to personality disorders includingbut not limited to the following types: paranoid, antisocial, avoidantbehavior, borderline personality disorders, dependent, histronic,narcissistic, obsessive-compulsive, schizoid, and schizotypal.

The invention also provides an agent that modulates a behavior which isassociated with gene disruption. In one aspect, the agent is an agonistor antagonist of the phenotype associated with a disruption of a genewhich encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. In yet another aspect, theagent is an agonist or antagonist of a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet anotheraspect, the agonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody. In still another aspect, the antagonist agent is ananti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a method of identifying an agent thatameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormalityassociated with a disruption in the gene which encodes for a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) administering a test agent to said non-human transgenic animal; and

(c) determining whether the test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality associated with the gene disruption in thenon-human transgenic animal.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities the retinalabnormalities are consistent with retinal dysplasia, variousretinopathies, including retinopathy of prematurity, retrolentalfibroplasia, neovascular glaucoma, age-related macular degeneration,diabetic macular edema, corneal neovascularization, corneal graftneovascularization, corneal graft rejection, retinal/choroidalneovascularization, neovascularization of the angle (rubeosis), ocularneovascular disease, vascular restenosis, arteriovenous malformations(AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,retinal artery obstruction or occlusion; retinal degeneration causingsecondary atrophy of the retinal vasculature, retinitis pigmentosa,macular dystrophies, Stargardt's disease, congenital stationary nightblindness, choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism, or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety-like response during open fieldactivity testing; abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; increased exploratoryactivity during open-field testing; increased stress inducedhyperthermia; enhanced motor coordination during inverted screentesting; impaired motor coordination during inverted screen testing;increase in retinal artery tortuosity; retinal degeneration marked byattenuated retinal vessels; opthamological abnormalities; increased meansystolic blood pressure; increased mean fasting serum glucose levels;decreased mean serum glucose levels; increased mean serum cholesterollevels; increased mean serum triglyceride levels; decreased mean serumcholesterol levels; decreased mean serum triglyceride levels; enhancedglucose tolerance; impaired glucose tolerance; increased mean seruminsulin levels; decreased mean serum insulin levels; increased uric acidlevels; decreased serum phosphate levels; increased alkaline phosphataselevels and increased alanine amino transferase levels; liver disease;increased mean percentage of CD25+ in both spleen and lymph nodes;decreased mean percentage of natural killer cells; decreased meanpercentage of CD21HiCD23Med cells in spleen and lymph nodes; increasedmean percentage of CD4 cells and decreased mean percentage of B cells;increased mean percentage of CD8+ cells; decreased mean percentage ofeosinophils; decreased mean serum IgG1 response to an ovalbuminchallenge; decreased mean serum IgG2a response to an ovalbuminchallenge; increased mean serum IgG1 response to an ovalbumin challenge;increased mean serum IgG2a response to an ovalbumin challenge; increasedmean serum MCP-1 response to a LPS challenge; increased mean serumTNF-alpha response to a LPS challenge; decreased mean serum MCP-1response to a LPS challenge; decreased mean serum IL-6 response to a LPSchallenge; decreased TNF-alpha response to a LPS challenge; increasedmean serum IL6 response to a LPS challenge; increased mean plateletcounts; decreased mean total white blood cell (WBC) counts; decreasedabsolute lymphocyte counts; decreased absolute monocyte counts;decreased skin fibroblast proliferation; increased skin fibroblastproliferation; increased mean percent of total body fat and total fatmass; increased mean body weight; increased mean body length; increasedorgan weights; increased total tissue mass (TTM); increased lean bodymass (LBM); increased bone mineral density (BMD) in total body, femurand vertebrae; increased bone mineral content (BMC) in total body, femurand vertebrae; increased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; increased mean femoral midshaft corticalthickness and cross-sectional area; increased mean vertebral trabecularbone volume, number and connectivity density; decreased mean percent oftotal body fat and total fat mass; decreased mean body weight; decreasedmean body length; decreased total tissue mass (TTM); decreased lean bodymass (LBM); decreased bone mineral density (BMD) in total body, femurand vertebrae; decreased bone mineral content (BMC) in total body, femurand vertebrae; decreased volumetric bone mineral density (vBMD) in totalbody, femur and vertebrae; decreased mean femoral midshaft corticalthickness and cross-sectional area; decreased mean vertebral trabecularbone volume, number and connectivity density; severe depletion ofabdominal and subcutaneous body fat deposits; decreased organ weights;growth retardation; hydrocephalus; sebaceous gland hyperplasia andgrowth retardation; apoptosis of olfactory neuroepithelial cells;lymphoid hyperplasia and tissue inflammation; development abnormalities;male infertility; growth retardation with reduced viability; andembryonic lethality. The invention also provides an agent thatameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormalitywhich is associated with gene disruption. In one aspect, the agent is anagonist or antagonist of the phenotype associated with a disruption of agene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. In yet another aspect, theagent is an agonist or antagonist of a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet anotheraspect, the agonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody. In still another aspect, the antagonist agent is ananti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a therapeutic agent for the treatment of aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality.

The invention also provides a method of identifying an agent thatmodulates the expression of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, the method comprising:

(a) contacting a test agent with a host cell expressing a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide; and

(b) determining whether the test agent modulates the expression of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide by the host cell.

The invention also provides an agent that modulates the expression of aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide. In one aspect, the agent is an agonist or antagonistof the phenotype associated with a disruption of a gene which encodesfor a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. In yet another aspect, the agent is anagonist or antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. In yet another aspect, theagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. Instill another aspect, the antagonist agent is an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a method of evaluating a therapeutic agentcapable of affecting a condition associated with a disruption of a genewhich encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as acondition resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) evaluating the effects of the test agent on the identified conditionassociated with gene disruption in the non-human transgenic animal.

In one aspect, the condition is a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.

The invention also provides a therapeutic agent which is capable ofaffecting a condition associated with gene disruption. In one aspect,the agent is an agonist or antagonist of the phenotype associated with adisruption of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet anotheraspect, the agent is an agonist or antagonist of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. Inyet another aspect, the agonist agent is an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody. In still another aspect, the antagonist agentis an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a pharmaceutical composition comprising atherapeutic agent capable of affecting the condition associated withgene disruption.

The invention also provides a method of treating or preventing orameliorating a neurological disorder; cardiovascular, endothelial orangiogenic disorder; immunological disorder; oncological disorder; bonemetabolic abnormality or disorder, or embryonic lethality associatedwith the disruption of a gene which encodes for a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide,the method comprising administering to a subject in need of suchtreatment whom may already have the disorder, or may be prone to havethe disorder or may be in whom the disorder is to be prevented, atherapeutically effective amount of a therapeutic agent, or agonists orantagonists thereof, thereby effectively treating or preventing orameliorating said disorder or disease.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect the therapeutic agent is an agonist or antagonist ofthe phenotype associated with a disruption of a gene which encodes for aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide. In yet another aspect, the agent is an agonist orantagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. In yet another aspect, the agonist agentis an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. In still anotheraspect, the antagonist agent is an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody.

The invention also provides a method of identifying an agent thatameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormalityassociated with a disruption in the gene which encodes for a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, the method comprising:

(a) providing a non-human transgenic animal cell culture, each cell ofsaid culture comprising a disruption of the gene which encodes for aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide;

(b) administering a test agent to said cell culture; and

(c) determining whether the test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in said culture. In yet another aspect, theneurological disorder is an increased anxiety-like response during openfield activity testing. In yet another aspect, the neurological disorderis a decreased anxiety-like response during open field activity testing.In yet another aspect, the neurological disorder is an abnormalcircadian rhythm during home-cage activity testing. In yet anotheraspect, the neurological disorder is an enhanced motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder is impaired motor coordination during inverted screen testing.In yet another aspect, the neurological disorder includes depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Such neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, socialanxiety, autism, specific phobia, substance-induced anxiety disorder,acute alcohol withdrawal, obsessive compulsive disorder, agoraphobia,monopolar disorders, bipolar disorder I or II, bipolar disorder nototherwise specified, cyclothymic disorder, depressive disorder, majordepressive disorder, mood disorder, substance-induced mood disorder,enhancement of cognitive function, loss of cognitive function associatedwith but not limited to Alzheimer's disease, stroke, or traumatic injuryto the brain, seizures resulting from disease or injury including butnot limited to epilepsy, learning disorders/disabilities, cerebralpalsy. In addition, anxiety disorders may apply to personality disordersincluding but not limited to the following types: paranoid, antisocial,avoidant behavior, borderline personality disorders, dependent,histronic, narcissistic, obsessive-compulsive, schizoid, andschizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

The invention also provides an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality which is associated with genedisruption in said culture. In one aspect, the agent is an agonist orantagonist of the phenotype associated with a disruption of a gene whichencodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. In yet another aspect, the agent is anagonist or antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. In yet another aspect, theagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. Instill another aspect, the antagonist agent is an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody.

The invention also provides a method of modulating a phenotypeassociated with a disruption of a gene which encodes for a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, the method comprising administering to a subject whom mayalready have the phenotype, or may be prone to have the phenotype or maybe in whom the phenotype is to be prevented, an effective amount of anagent identified as modulating said phenotype, or agonists orantagonists thereof, thereby effectively modulating the phenotype.

The invention also provides a method of modulating a physiologicalcharacteristic associated with a disruption of a gene which encodes fora PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, the method comprising administering to asubject whom may already exhibit the physiological characteristic, ormay be prone to exhibit the physiological characteristic or may be inwhom the physiological characteristic is to be prevented, an effectiveamount of an agent identified as modulating said physiologicalcharacteristic, or agonists or antagonists thereof, thereby effectivelymodulating the physiological characteristic.

The invention also provides a method of modulating a behavior associatedwith a disruption of a gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the methodcomprising administering to a subject whom may already exhibit thebehavior, or may be prone to exhibit the behavior or may be in whom theexhibited behavior is to be prevented, an effective amount of an agentidentified as modulating said behavior, or agonists or antagoniststhereof, thereby effectively modulating the behavior.

The invention also provides a method of modulating the expression of aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide, the method comprising administering to a host cellexpressing said PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, an effective amount of an agentidentified as modulating said expression, or agonists or antagoniststhereof, thereby effectively modulating the expression of saidpolypeptide.

The invention also provides a method of modulating a conditionassociated with a disruption of a gene which encodes for a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, the method comprising administering to a subject whom mayhave the condition, or may be prone to have the condition or may be inwhom the condition is to be prevented, a therapeutically effectiveamount of a therapeutic agent identified as modulating said condition,or agonists or antagonists thereof, thereby effectively modulating thecondition.

The invention also provides a method of treating or preventing orameliorating a neurological disorder; cardiovascular, endothelial orangiogenic disorder; immunological disorder; oncological disorder; bonemetabolic abnormality or disorder, or embryonic lethality associatedwith the disruption of a gene which encodes for a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide,the method comprising administering to a non-human transgenic animalcell culture, each cell of said culture comprising a disruption of thegene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, an effective amount of anagent identified as treating or preventing or ameliorating saiddisorder, or agonists or antagonists thereof, thereby effectivelytreating or preventing or ameliorating said disorder.

B. Further Embodiments

In yet further embodiments, the invention is directed to the followingset of potential claims for this application:

-   A method of identifying a phenotype associated with a disruption of    a gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,    PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,    PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,    PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,    PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779    polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal; and

(c) comparing the measured physiological characteristic with that of agender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal.

-   The method of Claim 1, wherein the non-human transgenic animal is    heterozygous for the disruption of a gene which encodes for a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   The method of Claim 1, wherein the phenotype exhibited by the    non-human transgenic animal as compared with gender matched    wild-type littermates is at least one of the following: a    neurological disorder; a cardiovascular, endothelial or angiogenic    disorder; an eye abnormality; an immunological disorder; an    oncological disorder; a bone metabolic abnormality or disorder; a    lipid metabolic disorder; or a developmental abnormality.-   4. The method of Claim 3, wherein the neurological disorder is an    increased anxiety-like response during open field activity testing.-   5. The method of Claim 3, wherein the neurological disorder is a    decreased anxiety-like response during open field activity testing.-   6. The method of Claim 3, wherein the neurological disorder is an    abnormal circadian rhythm during home-cage activity testing.-   7. The method of Claim 3, wherein the neurological disorder is an    enhanced motor coordination during inverted screen testing.-   8. The method of Claim 3, wherein the neurological disorder is an    impaired motor coordination during inverted screen testing.-   9. The method of Claim 3, wherein the neurological disorder is    depression, generalized anxiety disorders, attention deficit    disorder, sleep disorder, hyperactivity disorder, obsessive    compulsive disorder, schizophrenia, cognitive disorders,    hyperalgesia or sensory disorders.-   10. The method of Claim 3, wherein the eye abnormality is a retinal    abnormality.-   11. The method of Claim 3, wherein the eye abnormality is consistent    with vision problems or blindness.-   12. The method of Claim 10, wherein the retinal abnormality is    consistent with retinitis pigmentosa.-   13. The method of Claim 10, wherein the retinal abnormality is    characterized by retinal degeneration or retinal dysplasia.-   14. The method of Claim 10, wherein the retinal abnormality is    consistent with retinal dysplasia, various retinopathies, including    retinopathy of prematurity, retrolental fibroplasia, neovascular    glaucoma, age-related macular degeneration, diabetic macular edema,    corneal neovascularization, corneal graft neovascularization,    corneal graft rejection, retinal/choroidal neovascularization,    neovascularization of the angle (rubeosis), ocular neovascular    disease, vascular restenosis, arteriovenous malformations (AVM),    meningioma, hemangioma, angiofibroma, thyroid hyperplasias    (including Grave's disease), corneal and other tissue    transplantation, retinal artery obstruction or occlusion; retinal    degeneration causing secondary atrophy of the retinal vasculature,    retinitis pigmentosa, macular dystrophies, Stargardt's disease,    congenital stationary night blindness, choroideremia, gyrate    atrophy, Leber's congenital amaurosis, retinoschisis disorders,    Wagner's syndrome, Usher syndromes, Zellweger syndrome,    Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl    syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's    syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird    syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,    Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,    Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,    olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler    syndrome, carotinemeia, cystinosis, Wolfram syndrome,    Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia    pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,    or mannosidosis.-   15. The method of Claim 3, wherein the eye abnormality is a    cataract.-   16. The method of Claim 15, wherein the cataract is consistent with    systemic diseases such as human Down's syndrome, Hallerman-Streiff    syndrome, Lowe syndrome, galactosemia, Marfan syndrome, Trismoy    13-15, Alport syndrome, myotonic dystrophy, Fabry disease,    hypoparathroidism or Conradi syndrome.-   17. The method of Claim 3, wherein the developmental abnormality    comprises embryonic lethality or reduced viability.-   18. The method of Claim 3, wherein the cardiovascular, endothelial    or angiogenic disorders are arterial diseases, such as diabetes    mellitus; papilledema; optic atrophy; atherosclerosis; angina;    myocardial infarctions such as acute myocardial infarctions, cardiac    hypertrophy, and heart failure such as congestive heart failure;    hypertension; inflammatory vasculitides; Reynaud's disease and    Reynaud's phenomenon; aneurysms and arterial restenosis; venous and    lymphatic disorders such as thrombophlebitis, lymphangitis, and    lymphedema; peripheral vascular disease; cancer such as vascular    tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,    telangiectasia, bacillary angiomatosis, hemangioendothelioma,    angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,    and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,    burns, and other injured tissue, implant fixation, scarring;    ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular    disease; renal diseases such as acute renal failure, or    osteoporosis.-   19. The method of Claim 3, wherein the immunological disorders are    systemic lupus erythematosis; rheumatoid arthritis; juvenile chronic    arthritis; spondyloarthropathies; systemic sclerosis (scleroderma);    idiopathic inflammatory myopathies (dermatomyositis, polymyositis);    Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmune    hemolytic anemia (immune pancytopenia, paroxysmal nocturnal    hemoglobinuria); autoimmune thrombocytopenia (idiopathic    thrombocytopenic purpura, immune-mediated thrombocytopenia);    thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile    lymphocytic thyroiditis, atrophic thyroiditis); diabetes mellitus;    immune-mediated renal disease (glomerulonephritis,    tubulointerstitial nephritis); demyelinating diseases of the central    and peripheral nervous systems such as multiple sclerosis,    idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome,    and chronic inflammatory demyelinating polyneuropathy; hepatobiliary    diseases such as infectious hepatitis (hepatitis A, B, C, D, E and    other non-hepatotropic viruses), autoimmune chronic active    hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and    sclerosing cholangitis; inflammatory bowel disease (ulcerative    colitis: Crohn's disease); gluten-sensitive enteropathy, and    Whipple's disease; autoimmune or immune-mediated skin diseases    including bullous skin diseases, erythema multiforme and contact    dermatitis, psoriasis; allergic diseases such as asthma, allergic    rhinitis, atopic dermatitis, food hypersensitivity and urticaria;    immunologic diseases of the lung such as eosinophilic pneumonia,    idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; or    transplantation associated diseases including graft rejection and    graft-versus-host disease.-   20. The method of Claim 3, wherein the bone metabolic abnormality or    disorder is arthritis, osteoporosis or osteopetrosis.-   21. The method of Claim 1, wherein the non-human transgenic animal    exhibits at least one of the following physiological characteristics    compared with gender matched wild-type littermates: increased    anxiety-like response during open field testing; decreased    anxiety-like response during open field activity testing; abnormal    circadian rhythm during home-cage activity testing including    decreased ambulatory counts; increased exploratory activity during    open-field testing; increased stress induced hyperthermia; enhanced    motor coordination during inverted screen testing; impaired motor    coordination during inverted screen testing; increase in retinal    artery tortuosity; retinal degeneration marked by attenuated retinal    vessels; opthamological abnormalities; increased mean systolic blood    pressure; increased mean fasting serum glucose levels; decreased    mean serum glucose levels; increased mean serum cholesterol levels;    increased mean serum triglyceride levels; decreased mean serum    cholesterol levels; decreased mean serum triglyceride levels;    enhanced glucose tolerance; impaired glucose tolerance; increased    mean serum insulin levels; decreased mean serum insulin levels;    increased uric acid levels; decreased serum phosphate levels;    increased alkaline phosphatase levels and increased alanine amino    transferase levels; liver disease; increased mean percentage of    CD25+ in both spleen and lymph nodes; decreased mean percentage of    natural killer cells; decreased mean percentage of CD21HiCD23Med    cells in spleen and lymph nodes; increased mean percentage of CD4    cells and decreased mean percentage of B cells; increased mean    percentage of CD8+ cells; decreased mean percentage of eosinophils;    decreased mean serum IgG1 response to an ovalbumin challenge;    decreased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum IgG1 response to an ovalbumin challenge;    increased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum MCP-1 response to a LPS challenge; increased    mean serum TNF-alpha response to a LPS challenge; decreased mean    serum MCP-1 response to a LPS challenge; decreased mean serum IL-6    response to a LPS challenge; decreased TNF-alpha response to a LPS    challenge; increased mean serum IL6 response to a LPS challenge;    increased mean platelet counts; decreased mean total white blood    cell (WBC) counts; decreased absolute lymphocyte counts; decreased    absolute monocyte counts; decreased skin fibroblast proliferation;    increased skin fibroblast proliferation; increased mean percent of    total body fat and total fat mass; increased mean body weight;    increased mean body length; increased organ weights; increased total    tissue mass (TTM); increased lean body mass (LBM); increased bone    mineral density (BMD) in total body, femur and vertebrae; increased    bone mineral content (BMC) in total body, femur and vertebrae;    increased volumetric bone mineral density (vBMD) in total body,    femur and vertebrae; increased mean femoral midshaft cortical    thickness and cross-sectional area; increased mean vertebral    trabecular bone volume, number and connectivity density; decreased    mean percent of total body fat and total fat mass; decreased mean    body weight; decreased mean body length; decreased total tissue mass    (TTM); decreased lean body mass (LBM); decreased bone mineral    density (BMD) in total body, femur and vertebrae; decreased bone    mineral content (BMC) in total body, femur and vertebrae; decreased    volumetric bone mineral density (vBMD) in total body, femur and    vertebrae; decreased mean femoral midshaft cortical thickness and    cross-sectional area; decreased mean vertebral trabecular bone    volume, number and connectivity density; severe depletion of    abdominal and subcutaneous body fat deposits; decreased organ    weights; growth retardation; hydrocephalus; sebaceous gland    hyperplasia and growth retardation; apoptosis of olfactory    neuroepithelial cells; lymphoid hyperplasia and tissue inflammation;    development abnormalities; male infertility; growth retardation with    reduced viability; and embryonic lethality.-   22. An isolated cell derived from a non-human transgenic animal    whose genome comprises a disruption of the gene which encodes for a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   23. The isolated cell of Claim 22 which is a murine cell.-   24. The isolated cell of Claim 23, wherein the murine cell is an    embryonic stem cell.-   25. The isolated cell of Claim 22, wherein the non-human transgenic    animal exhibits at least one of the following phenotypes compared    with gender matched wild-type littermates: a neurological disorder;    a cardiovascular, endothelial or angiogenic disorder; an eye    abnormality; an immunological disorder; an oncological disorder; a    bone metabolic abnormality or disorder; a lipid metabolic disorder;    or a developmental abnormality.-   26. A method of identifying an agent that modulates a phenotype    associated with a disruption of a gene which encodes for a PRO196,    PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,    PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,    PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,    PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,    PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the test agent modulates the identifiedphenotype associated with gene disruption in the non-human transgenicanimal.

-   27. The method of Claim 26, wherein the phenotype associated with    the gene disruption comprises a neurological disorder; a    cardiovascular, endothelial or angiogenic disorder; an eye    abnormality; an immunological disorder; an oncological disorder; a    bone metabolic abnormality or disorder; a lipid metabolic disorder;    or a developmental abnormality.-   28. The method of Claim 27, wherein the neurological disorder is an    increased anxiety-like response during open field activity testing.-   29. The method of Claim 27, wherein the neurological disorder is a    decreased anxiety-like response during open field activity testing.-   30. The method of Claim 27, wherein the neurological disorder is an    abnormal circadian rhythm during home-cage activity testing.-   31. The method of Claim 27, wherein the neurological disorder is an    enhanced motor coordination during inverted screen testing.-   32. The method of Claim 27, wherein the neurological disorder is an    impaired motor coordination during inverted screen testing.-   33. The method of Claim 27, wherein the neurological disorder is    depression, generalized anxiety disorders, attention deficit    disorder, sleep disorder, hyperactivity disorder, obsessive    compulsive disorder, schizophrenia, cognitive disorders,    hyperalgesia or sensory disorders.-   34. The method of Claim 27, wherein the eye abnormality is a retinal    abnormality.-   35. The method of Claim 27, wherein the eye abnormality is    consistent with vision problems or blindness.-   36. The method of Claim 34, wherein the retinal abnormality is    consistent with retinitis pigmentosa.-   37. The method of Claim 34, wherein the retinal abnormality is    characterized by retinal degeneration or retinal dysplasia.-   38. The method of Claim 34, wherein the retinal abnormality is    consistent with retinal dysplasia, various retinopathies, including    retinopathy of prematurity, retrolental fibroplasia, neovascular    glaucoma, age-related macular degeneration, diabetic macular edema,    corneal neovascularization, corneal graft neovascularization,    corneal graft rejection, retinal/choroidal neovascularization,    neovascularization of the angle (rubeosis), ocular neovascular    disease, vascular restenosis, arteriovenous malformations (AVM),    meningioma, hemangioma, angiofibroma, thyroid hyperplasias    (including Grave's disease), corneal and other tissue    transplantation, retinal artery obstruction or occlusion; retinal    degeneration causing secondary atrophy of the retinal vasculature,    retinitis pigmentosa, macular dystrophies, Stargardt's disease,    congenital stationary night blindness, choroideremia, gyrate    atrophy, Leber's congenital amaurosis, retinoschisis disorders,    Wagner's syndrome, Usher syndromes, Zellweger syndrome,    Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl    syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's    syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird    syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,    Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,    Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,    olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler    syndrome, carotinemeia, cystinosis, Wolfram syndrome,    Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia    pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,    or mannosidosis.-   39. The method of Claim 27, wherein the eye abnormality is a    cataract.-   40. The method of Claim 39, wherein the cataract is consistent with    systemic diseases such as human Down's syndrome, Hallerman-Streiff    syndrome, Lowe syndrome, galactosemia, Marfan syndrome, Trismoy    13-15, Alport syndrome, myotonic dystrophy, Fabry disease,    hypoparathroidism or Conradi syndrome.-   41. The method of Claim 27, wherein the developmental abnormality    comprises embryonic lethality or reduced viability.-   42. The method of Claim 27, wherein the cardiovascular, endothelial    or angiogenic disorders are arterial diseases, such as diabetes    mellitus; papilledema; optic atrophy; atherosclerosis; angina;    myocardial infarctions such as acute myocardial infarctions, cardiac    hypertrophy, and heart failure such as congestive heart failure;    hypertension; inflammatory vasculitides; Reynaud's disease and    Reynaud's phenomenon; aneurysms and arterial restenosis; venous and    lymphatic disorders such as thrombophlebitis, lymphangitis, and    lymphedema; peripheral vascular disease; cancer such as vascular    tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,    telangiectasia, bacillary angiomatosis, hemangioendothelioma,    angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,    and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,    burns, and other injured tissue, implant fixation, scarring;    ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular    disease; renal diseases such as acute renal failure, or    osteoporosis.-   43. The method of Claim 27, wherein the immunological disorders are    systemic lupus erythematosis; rheumatoid arthritis; juvenile chronic    arthritis; spondyloarthropathies; systemic sclerosis (scleroderma);    idiopathic inflammatory myopathies (dermatomyositis, polymyositis);    Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmune    hemolytic anemia (immune pancytopenia, paroxysmal nocturnal    hemoglobinuria); autoimmune thrombocytopenia (idiopathic    thrombocytopenic purpura, immune-mediated thrombocytopenia);    thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile    lymphocytic thyroiditis, atrophic thyroiditis); diabetes mellitus;    immune-mediated renal disease (glomerulonephritis,    tubulointerstitial nephritis); demyelinating diseases of the central    and peripheral nervous systems such as multiple sclerosis,    idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome,    and chronic inflammatory demyelinating polyneuropathy; hepatobiliary    diseases such as infectious hepatitis (hepatitis A, B, C, D, E and    other non-hepatotropic viruses), autoimmune chronic active    hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and    sclerosing cholangitis; inflammatory bowel disease (ulcerative    colitis: Crohn's disease); gluten-sensitive enteropathy, and    Whipple's disease; autoimmune or immune-mediated skin diseases    including bullous skin diseases, erythema multiforme and contact    dermatitis, psoriasis; allergic diseases such as asthma, allergic    rhinitis, atopic dermatitis, food hypersensitivity and urticaria;    immunologic diseases of the lung such as eosinophilic pneumonia,    idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; or    transplantation-associated diseases including graft rejection and    graft-versus-host disease.-   44. The method of Claim 27, wherein said bone metabolic abnormality    or disorder is arthritis, osteoporosis or osteopetrosis.-   45. The method of Claim 26, wherein the non-human transgenic animal    exhibits at least one of the following physiological characteristics    compared with gender matched wild-type littermates: increased    anxiety-like response during open field testing; decreased    anxiety-like response during open field activity testing; abnormal    circadian rhythm during home-cage activity testing including    decreased ambulatory counts; increased exploratory activity during    open-field testing; increased stress induced hyperthermia; enhanced    motor coordination during inverted screen testing; impaired motor    coordination during inverted screen testing; increase in retinal    artery tortuosity; retinal degeneration marked by attenuated retinal    vessels; opthamological abnormalities; increased mean systolic blood    pressure; increased mean fasting serum glucose levels; decreased    mean serum glucose levels; increased mean serum cholesterol levels;    increased mean serum triglyceride levels; decreased mean serum    cholesterol levels; decreased mean serum triglyceride levels;    enhanced glucose tolerance; impaired glucose tolerance; increased    mean serum insulin levels; decreased mean serum insulin levels;    increased uric acid levels; decreased serum phosphate levels;    increased alkaline phosphatase levels and increased alanine amino    transferase levels; liver disease; increased mean percentage of    CD25+ in both spleen and lymph nodes; decreased mean percentage of    natural killer cells; decreased mean percentage of CD21HiCD23Med    cells in spleen and lymph nodes; increased mean percentage of CD4    cells and decreased mean percentage of B cells; increased mean    percentage of CD8+ cells; decreased mean percentage of eosinophils;    decreased mean serum IgG1 response to an ovalbumin challenge;    decreased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum IgG1 response to an ovalbumin challenge;    increased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum MCP-1 response to a LPS challenge; increased    mean serum TNF-alpha response to a LPS challenge; decreased mean    serum MCP-1 response to a LPS challenge; decreased mean serum IL-6    response to a LPS challenge; decreased TNF-alpha response to a LPS    challenge; increased mean serum IL6 response to a LPS challenge;    increased mean platelet counts; decreased mean total white blood    cell (WBC) counts; decreased absolute lymphocyte counts; decreased    absolute monocyte counts; decreased skin fibroblast proliferation;    increased skin fibroblast proliferation; increased mean percent of    total body fat and total fat mass; increased mean body weight;    increased mean body length; increased organ weights; increased total    tissue mass (TTM); increased lean body mass (LBM); increased bone    mineral density (BMD) in total body, femur and vertebrae; increased    bone mineral content (BMC) in total body, femur and vertebrae;    increased volumetric bone mineral density (vBMD) in total body,    femur and vertebrae; increased mean femoral midshaft cortical    thickness and cross-sectional area; increased mean vertebral    trabecular bone volume, number and connectivity density; decreased    mean percent of total body fat and total fat mass; decreased mean    body weight; decreased mean body length; decreased total tissue mass    (TTM); decreased lean body mass (LBM); decreased bone mineral    density (BMD) in total body, femur and vertebrae; decreased bone    mineral content (BMC) in total body, femur and vertebrae; decreased    volumetric bone mineral density (vBMD) in total body, femur and    vertebrae; decreased mean femoral midshaft cortical thickness and    cross-sectional area; decreased mean vertebral trabecular bone    volume, number and connectivity density; severe depletion of    abdominal and subcutaneous body fat deposits; decreased organ    weights; growth retardation; hydrocephalus; sebaceous gland    hyperplasia and growth retardation; apoptosis of olfactory    neuroepithelial cells; lymphoid hyperplasia and tissue inflammation;    development abnormalities; male infertility; growth retardation with    reduced viability; and embryonic lethality.-   46. An agent identified by the method of Claim 26.-   47. The agent of Claim 46 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   48. The agent of Claim 47, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   49. The agent of Claim 47, wherein the antagonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   50. A method of identifying an agent that modulates a physiological    characteristic associated with a disruption of the gene which    encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,    PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,    PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,    PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,    PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the    method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic exhibited by the non-humantransgenic animal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic exhibited by the non-human transgenic animal that differsfrom the physiological characteristic exhibited by the wild-type animalis identified as a physiological characteristic associated with genedisruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the physiological characteristic associated withgene disruption is modulated.

-   51. The method of Claim 50, wherein the non-human transgenic animal    exhibits at least one of the following physiological characteristics    compared with gender matched wild-type littermates: increased    anxiety-like response during open field testing; decreased    anxiety-like response during open field activity testing; abnormal    circadian rhythm during home-cage activity testing including    decreased ambulatory counts; increased exploratory activity during    open-field testing; increased stress induced hyperthermia; enhanced    motor coordination during inverted screen testing; impaired motor    coordination during inverted screen testing; increase in retinal    artery tortuosity; retinal degeneration marked by attenuated retinal    vessels; opthamological abnormalities; increased mean systolic blood    pressure; increased mean fasting serum glucose levels; decreased    mean serum glucose levels; increased mean serum cholesterol levels;    increased mean serum triglyceride levels; decreased mean serum    cholesterol levels; decreased mean serum triglyceride levels;    enhanced glucose tolerance; impaired glucose tolerance; increased    mean serum insulin levels; decreased mean serum insulin levels;    increased uric acid levels; decreased serum phosphate levels;    increased alkaline phosphatase levels and increased alanine amino    transferase levels; liver disease; increased mean percentage of    CD25+ in both spleen and lymph nodes; decreased mean percentage of    natural killer cells; decreased mean percentage of CD21HiCD23Med    cells in spleen and lymph nodes; increased mean percentage of CD4    cells and decreased mean percentage of B cells; increased mean    percentage of CD8+ cells; decreased mean percentage of eosinophils;    decreased mean serum IgG1 response to an ovalbumin challenge;    decreased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum IgG1 response to an ovalbumin challenge;    increased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum MCP-1 response to a LPS challenge; increased    mean serum TNF-alpha response to a LPS challenge; decreased mean    serum MCP-1 response to a LPS challenge; decreased mean serum IL-6    response to a LPS challenge; decreased TNF-alpha response to a LPS    challenge; increased mean serum IL6 response to a LPS challenge;    increased mean platelet counts; decreased mean total white blood    cell (WBC) counts; decreased absolute lymphocyte counts; decreased    absolute monocyte counts; decreased skin fibroblast proliferation;    increased skin fibroblast proliferation; increased mean percent of    total body fat and total fat mass; increased mean body weight;    increased mean body length; increased organ weights; increased total    tissue mass (TTM); increased lean body mass (LBM); increased bone    mineral density (BMD) in total body, femur and vertebrae; increased    bone mineral content (BMC) in total body, femur and vertebrae;    increased volumetric bone mineral density (vBMD) in total body,    femur and vertebrae; increased mean femoral midshaft cortical    thickness and cross-sectional area; increased mean vertebral    trabecular bone volume, number and connectivity density; decreased    mean percent of total body fat and total fat mass; decreased mean    body weight; decreased mean body length; decreased total tissue mass    (TTM); decreased lean body mass (LBM); decreased bone mineral    density (BMD) in total body, femur and vertebrae; decreased bone    mineral content (BMC) in total body, femur and vertebrae; decreased    volumetric bone mineral density (vBMD) in total body, femur and    vertebrae; decreased mean femoral midshaft cortical thickness and    cross-sectional area; decreased mean vertebral trabecular bone    volume, number and connectivity density; severe depletion of    abdominal and subcutaneous body fat deposits; decreased organ    weights; growth retardation; hydrocephalus; sebaceous gland    hyperplasia and growth retardation; apoptosis of olfactory    neuroepithelial cells; lymphoid hyperplasia and tissue inflammation;    development abnormalities; male infertility; growth retardation with    reduced viability; and embryonic lethality.-   52. An agent identified by the method of Claim 50.-   53. The agent of Claim 52 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   54. The agent of Claim 53, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   55. The agent of Claim 53, wherein the antagonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   56. A method of identifying an agent which modulates a behavior    associated with a disruption of the gene which encodes for a PRO196,    PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,    PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,    PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,    PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,    PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) observing the behavior exhibited by the non-human transgenic animalof (a);

(c) comparing the observed behavior of (b) with that of a gender matchedwild-type animal, wherein the observed behavior exhibited by thenon-human transgenic animal that differs from the observed behaviorexhibited by the wild-type animal is identified as a behavior associatedwith gene disruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the agent modulates the behavior associated withgene disruption.

-   57. The method of Claim 56, wherein the behavior is an increased    anxiety-like response during open field activity testing.-   58. The method of Claim 56, wherein the behavior is a decreased    anxiety-like response during open field activity testing.-   59. The method of Claim 56, wherein the behavior is an abnormal    circadian rhythm during home-cage activity testing.-   60. The method of Claim 56, wherein the behavior is an enhanced    motor coordination during inverted screen testing.-   61. The method of Claim 56, wherein the behavior is an impaired    motor coordination during inverted screen testing.-   62. The method of Claim 56, wherein the behavior is depression,    generalized anxiety disorders, attention deficit disorder, sleep    disorder, hyperactivity disorder, obsessive compulsive disorder,    schizophrenia, cognitive disorders, hyperalgesia or sensory    disorders.-   63. An agent identified by the method of Claim 56.-   64. The agent of Claim 63 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   65. The agent of Claim 64, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   66. The agent of Claim 64, wherein the antagonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   67. A method of identifying an agent that ameliorates or modulates a    neurological disorder; a cardiovascular, endothelial or angiogenic    disorder; an eye abnormality; an immunological disorder; an    oncological disorder; a bone metabolic abnormality or disorder; a    lipid metabolic disorder; or a developmental abnormality associated    with a disruption in the gene which encodes for a PRO196, PRO217,    PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,    PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,    PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,    PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or    PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) administering a test agent to said non-human transgenic animal; and

(c) determining whether said test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in the non-human transgenic animal.

-   68. The method of Claim 67, wherein the neurological disorder is an    increased anxiety-like response during open field activity testing.-   69. The method of Claim 67, wherein the neurological disorder is a    decreased anxiety-like response during open field activity testing.-   70. The method of Claim 67, wherein the neurological disorder is an    abnormal circadian rhythm during home-cage activity testing.-   71. The method of Claim 67, wherein the neurological disorder is an    enhanced motor coordination during inverted screen testing.-   72. The method of Claim 67, wherein the neurological disorder is an    impaired motor coordination during inverted screen testing.-   73. The method of Claim 73, wherein the neurological disorder is    depression, generalized anxiety disorders, attention deficit    disorder, sleep disorder, hyperactivity disorder, obsessive    compulsive disorder, schizophrenia, cognitive disorders,    hyperalgesia or sensory disorders.-   74. The method of Claim 67, wherein the eye abnormality is a retinal    abnormality.-   75. The method of Claim 67, wherein the eye abnormality is    consistent with vision problems or blindness.-   76. The method of Claim 74, wherein the retinal abnormality is    consistent with retinitis pigmentosa.-   77. The method of Claim 74, wherein the retinal abnormality is    characterized by retinal degeneration or retinal dysplasia.-   78. The method of Claim 74, wherein the retinal abnormality is    consistent with retinal dysplasia, various retinopathies, including    retinopathy of prematurity, retrolental fibroplasia, neovascular    glaucoma, age-related macular degeneration, diabetic macular edema,    corneal neovascularization, corneal graft neovascularization,    corneal graft rejection, retinal/choroidal neovascularization,    neovascularization of the angle (rubeosis), ocular neovascular    disease, vascular restenosis, arteriovenous malformations (AVM),    meningioma, hemangioma, angiofibroma, thyroid hyperplasias    (including Grave's disease), corneal and other tissue    transplantation, retinal artery obstruction or occlusion; retinal    degeneration causing secondary atrophy of the retinal vasculature,    retinitis pigmentosa, macular dystrophies, Stargardt's disease,    congenital stationary night blindness, choroideremia, gyrate    atrophy, Leber's congenital amaurosis, retinoschisis disorders,    Wagner's syndrome, Usher syndromes, Zellweger syndrome,    Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl    syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's    syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird    syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,    Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,    Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,    olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler    syndrome, carotinemeia, cystinosis, Wolfram syndrome,    Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia    pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,    or mannosidosis.-   79. The method of Claim 67, wherein the eye abnormality is a    cataract.-   80. The method of Claim 79, wherein the cataract is a systemic    disease such as human Down's syndrome, Hallerman-Streiff syndrome,    Lowe syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport    syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or    Conradi syndrome.-   81. The method of Claim 67, wherein the developmental abnormality    comprises embryonic lethality or reduced viability.-   82. The method of Claim 67, wherein the cardiovascular, endothelial    or angiogenic disorders are arterial diseases, such as diabetes    mellitus; papilledema; optic atrophy; atherosclerosis; angina;    myocardial infarctions such as acute myocardial infarctions, cardiac    hypertrophy, and heart failure such as congestive heart failure;    hypertension; inflammatory vasculitides; Reynaud's disease and    Reynaud's phenomenon; aneurysms and arterial restenosis; venous and    lymphatic disorders such as thrombophlebitis, lymphangitis, and    lymphedema; peripheral vascular disease; cancer such as vascular    tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,    telangiectasia, bacillary angiomatosis, hemangioendothelioma,    angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,    and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,    burns, and other injured tissue, implant fixation, scarring;    ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular    disease; renal diseases such as acute renal failure, or    osteoporosis.-   83. The method of Claim 67, wherein the immunological disorders are    systemic lupus erythematosis; rheumatoid arthritis; juvenile chronic    arthritis; spondyloarthropathies; systemic sclerosis (scleroderma);    idiopathic inflammatory myopathies (dermatomyositis, polymyositis);    Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmune    hemolytic anemia (immune pancytopenia, paroxysmal nocturnal    hemoglobinuria); autoimmune thrombocytopenia (idiopathic    thrombocytopenic purpura, immune-mediated thrombocytopenia);    thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile    lymphocytic thyroiditis, atrophic thyroiditis); diabetes mellitus;    immune-mediated renal disease (glomerulonephritis,    tubulointerstitial nephritis); demyelinating diseases of the central    and peripheral nervous systems such as multiple sclerosis,    idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome,    and chronic inflammatory demyelinating polyneuropathy; hepatobiliary    diseases such as infectious hepatitis (hepatitis A, B, C, D, E and    other non-hepatotropic viruses), autoimmune chronic active    hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and    sclerosing cholangitis; inflammatory bowel disease (ulcerative    colitis: Crohn's disease); gluten-sensitive enteropathy, and    Whipple's disease; autoimmune or immune-mediated skin diseases    including bullous skin diseases, erythema multiforme and contact    dermatitis, psoriasis; allergic diseases such as asthma, allergic    rhinitis, atopic dermatitis, food hypersensitivity and urticaria;    immunologic diseases of the lung such as eosinophilic pneumonia,    idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; or    transplantation associated diseases including graft rejection and    graft-versus-host disease.-   84. The method of Claim 67, wherein said bone metabolic abnormality    or disorder is arthritis, osteoporosis or osteopetrosis.-   85. The method of Claim 67, wherein the non-human transgenic animal    exhibits at least one of the following physiological characteristics    compared with gender matched wild-type littermates: increased    anxiety-like response during open field testing; decreased    anxiety-like response during open field activity testing; abnormal    circadian rhythm during home-cage activity testing including    decreased ambulatory counts; increased exploratory activity during    open-field testing; increased stress induced hyperthermia; enhanced    motor coordination during inverted screen testing; impaired motor    coordination during inverted screen testing; increase in retinal    artery tortuosity; retinal degeneration marked by attenuated retinal    vessels; opthamological abnormalities; increased mean systolic blood    pressure; increased mean fasting serum glucose levels; decreased    mean serum glucose levels; increased mean serum cholesterol levels;    increased mean serum triglyceride levels; decreased mean serum    cholesterol levels; decreased mean serum triglyceride levels;    enhanced glucose tolerance; impaired glucose tolerance; increased    mean serum insulin levels; decreased mean serum insulin levels;    increased uric acid levels; decreased serum phosphate levels;    increased alkaline phosphatase levels and increased alanine amino    transferase levels; liver disease; increased mean percentage of    CD25+ in both spleen and lymph nodes; decreased mean percentage of    natural killer cells; decreased mean percentage of CD21HiCD23Med    cells in spleen and lymph nodes; increased mean percentage of CD4    cells and decreased mean percentage of B cells; increased mean    percentage of CD8+ cells; decreased mean percentage of eosinophils;    decreased mean serum IgG1 response to an ovalbumin challenge;    decreased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum IgG1 response to an ovalbumin challenge;    increased mean serum IgG2a response to an ovalbumin challenge;    increased mean serum MCP-1 response to a LPS challenge; increased    mean serum TNF-alpha response to a LPS challenge; decreased mean    serum MCP-1 response to a LPS challenge; decreased mean serum IL-6    response to a LPS challenge; decreased TNF-alpha response to a LPS    challenge; increased mean serum IL6 response to a LPS challenge;    increased mean platelet counts; decreased mean total white blood    cell (WBC) counts; decreased absolute lymphocyte counts; decreased    absolute monocyte counts; decreased skin fibroblast proliferation;    increased skin fibroblast proliferation; increased mean percent of    total body fat and total fat mass; increased mean body weight;    increased mean body length; increased organ weights; increased total    tissue mass (TTM); increased lean body mass (LBM); increased bone    mineral density (BMD) in total body, femur and vertebrae; increased    bone mineral content (BMC) in total body, femur and vertebrae;    increased volumetric bone mineral density (vBMD) in total body,    femur and vertebrae; increased mean femoral midshaft cortical    thickness and cross-sectional area; increased mean vertebral    trabecular bone volume, number and connectivity density; decreased    mean percent of total body fat and total fat mass; decreased mean    body weight; decreased mean body length; decreased total tissue mass    (TTM); decreased lean body mass (LBM); decreased bone mineral    density (BMD) in total body, femur and vertebrae; decreased bone    mineral content (BMC) in total body, femur and vertebrae; decreased    volumetric bone mineral density (vBMD) in total body, femur and    vertebrae; decreased mean femoral midshaft cortical thickness and    cross-sectional area; decreased mean vertebral trabecular bone    volume, number and connectivity density; severe depletion of    abdominal and subcutaneous body fat deposits; decreased organ    weights; growth retardation; hydrocephalus; sebaceous gland    hyperplasia and growth retardation; apoptosis of olfactory    neuroepithelial cells; lymphoid hyperplasia and tissue inflammation;    development abnormalities; male infertility; growth retardation with    reduced viability; and embryonic lethality.-   86. An agent identified by the method of Claim 67.-   87. The agent of Claim 86 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   88. The agent of Claim 87, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   89. The agent of Claim 87, wherein the antagonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   90. A therapeutic agent identified by the method of Claim 67.-   91. A method of identifying an agent that modulates the expression    of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide, the method comprising:

(a) contacting a test agent with a host cell expressing a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide; and

(b) determining whether the test agent modulates the expression of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide by the host cell.

-   92. An agent identified by the method of Claim 91.-   93. The agent of Claim 92 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   94. The agent of Claim 93, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   95. The agent of Claim 93, wherein the antagonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   96. A method of evaluating a therapeutic agent capable of affecting    a condition associated with a disruption of a gene which encodes for    a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as acondition resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) evaluating the effects of the test agent on the identified conditionassociated with gene disruption in the non-human transgenic animal.

-   97. The method of Claim 96, wherein the condition is a neurological    disorder; a cardiovascular, endothelial or angiogenic disorder; an    eye abnormality; an immunological disorder; an oncological disorder;    a bone metabolic abnormality or disorder; a lipid metabolic    disorder; or a developmental abnormality.-   98. A therapeutic agent identified by the method of Claim 96.-   99. The therapeutic agent of Claim 98 which is an agonist or    antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,    PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,    PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,    PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,    PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.-   100. The therapeutic agent of Claim 99, wherein the agonist is an    anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,    anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,    anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,    anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,    anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,    anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,    anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,    anti-PRO61709 or anti-PRO779 antibody.-   101. The therapeutic agent of Claim 99, wherein the antagonist is an    anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,    anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,    anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,    anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,    anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,    anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,    anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,    anti-PRO61709 or anti-PRO779 antibody.-   102. A pharmaceutical composition comprising the therapeutic agent    of Claim 98.-   103. A method of treating or preventing or ameliorating a    neurological disorder; cardiovascular, endothelial or angiogenic    disorder; immunological disorder; oncological disorder; bone    metabolic abnormality or disorder, or embryonic lethality associated    with the disruption of a gene which encodes for a PRO196, PRO217,    PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,    PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,    PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,    PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or    PRO779 polypeptide, the method comprising administering to a subject    in need of such treatment whom may already have the disorder, or may    be prone to have the disorder or may be in whom the disorder is to    be prevented, a therapeutically effective amount of the therapeutic    agent of Claim 94, or agonists or antagonists thereof, thereby    effectively treating or preventing or ameliorating said disorder.-   104. The method of Claim 103, wherein the neurological disorder is    an increased anxiety-like response during open field activity    testing.-   105. The method of Claim 103, wherein the neurological disorder is a    decreased anxiety-like response during open field activity testing.-   106. The method of Claim 103, wherein the neurological disorder is    an abnormal circadian rhythm during home-cage activity testing.-   107. The method of Claim 103, wherein the neurological disorder is    an enhanced motor coordination during inverted screen testing.-   108. The method of Claim 103, wherein the neurological disorder is    an impaired motor coordination during inverted screen testing.-   109. The method of Claim 103, wherein the neurological disorder is    depression, generalized anxiety disorders, attention deficit    disorder, sleep disorder, hyperactivity disorder, obsessive    compulsive disorder, schizophrenia, cognitive disorders,    hyperalgesia or sensory disorders.-   110. The method of Claim 103, wherein the eye abnormality is a    retinal abnormality.-   111. The method of Claim 103, wherein the eye abnormality is    consistent with vision problems or blindness.-   112. The method of Claim 110, wherein the retinal abnormality is    consistent with retinitis pigmentosa.-   113. The method of Claim 110, wherein the retinal abnormality is    characterized by retinal degeneration or retinal dysplasia.-   114. The method of Claim 110, wherein the retinal abnormality is    consistent with retinal dysplasia, various retinopathies, including    retinopathy of prematurity, retrolental fibroplasia, neovascular    glaucoma, age-related macular degeneration, diabetic macular edema,    corneal neovascularization, corneal graft neovascularization,    corneal graft rejection, retinal/choroidal neovascularization,    neovascularization of the angle (rubeosis), ocular neovascular    disease, vascular restenosis, arteriovenous malformations (AVM),    meningioma, hemangioma, angiofibroma, thyroid hyperplasias    (including Grave's disease), corneal and other tissue    transplantation, retinal artery obstruction or occlusion; retinal    degeneration causing secondary atrophy of the retinal vasculature,    retinitis pigmentosa, macular dystrophies, Stargardt's disease,    congenital stationary night blindness, choroideremia, gyrate    atrophy, Leber's congenital amaurosis, retinoschisis disorders,    Wagner's syndrome, Usher syndromes, Zellweger syndrome,    Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl    syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's    syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird    syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,    Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,    Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,    olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler    syndrome, carotinemeia, cystinosis, Wolfram syndrome,    Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia    pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,    or mannosidosis.-   115. The method of Claim 103, wherein the eye abnormality is a    cataract.-   116. The method of Claim 115, wherein the cataract is a systemic    disease such as human Down's syndrome, Hallerman-Streiff syndrome,    Lowe syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport    syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or    Conradi syndrome.-   117. The method of Claim 103, wherein the developmental abnormality    comprises embryonic lethality or reduced viability.-   118. The method of Claim 103, wherein the cardiovascular,    endothelial or angiogenic disorders are arterial diseases, such as    diabetes mellitus; papilledema; optic atrophy; atherosclerosis;    angina; myocardial infarctions such as acute myocardial infarctions,    cardiac hypertrophy, and heart failure such as congestive heart    failure; hypertension; inflammatory vasculitides; Reynaud's disease    and Reynaud's phenomenon; aneurysms and arterial restenosis; venous    and lymphatic disorders such as thrombophlebitis, lymphangitis, and    lymphedema; peripheral vascular disease; cancer such as vascular    tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,    telangiectasia, bacillary angiomatosis, hemangioendothelioma,    angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,    and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,    burns, and other injured tissue, implant fixation, scarring;    ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular    disease; renal diseases such as acute renal failure, or    osteoporosis.-   119. The method of Claim 103, wherein the immunological disorders    are systemic lupus erythematosis; rheumatoid arthritis; juvenile    chronic arthritis; spondyloarthropathies; systemic sclerosis    (scleroderma); idiopathic inflammatory myopathies (dermatomyositis,    polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;    autoimmune hemolytic anemia (immune pancytopenia, paroxysmal    nocturnal hemoglobinuria); autoimmune thrombocytopenia (idiopathic    thrombocytopenic purpura, immune-mediated thrombocytopenia);    thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile    lymphocytic thyroiditis, atrophic thyroiditis); diabetes mellitus;    immune-mediated renal disease (glomerulonephritis,    tubulointerstitial nephritis); demyelinating diseases of the central    and peripheral nervous systems such as multiple sclerosis,    idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome,    and chronic inflammatory demyelinating polyneuropathy; hepatobiliary    diseases such as infectious hepatitis (hepatitis A, B, C, D, E and    other non-hepatotropic viruses), autoimmune chronic active    hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and    sclerosing cholangitis; inflammatory bowel disease (ulcerative    colitis: Crohn's disease); gluten-sensitive enteropathy, and    Whipple's disease; autoimmune or immune-mediated skin diseases    including bullous skin diseases, erythema multiforme and contact    dermatitis, psoriasis; allergic diseases such as asthma, allergic    rhinitis, atopic dermatitis, food hypersensitivity and urticaria;    immunologic diseases of the lung such as eosinophilic pneumonia,    idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; or    transplantation associated diseases including graft rejection and    graft-versus-host disease.-   120. The method of Claim 103, wherein said bone metabolic    abnormality or disorder is arthritis, osteoporosis or osteopetrosis.-   121. A method of identifying an agent that ameliorates or modulates    a neurological disorder; a cardiovascular, endothelial or angiogenic    disorder; an eye abnormality; an immunological disorder; an    oncological disorder; a bone metabolic abnormality or disorder; a    lipid metabolic disorder; or a developmental abnormality associated    with a disruption in the gene which encodes for a PRO196, PRO217,    PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,    PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,    PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,    PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or    PRO779 polypeptide, the method comprising:

(a) providing a non-human transgenic animal cell culture, each cell ofsaid culture comprising a disruption of the gene which encodes for aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide;

(b) administering a test agent to said cell culture; and

(c) determining whether said test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in said cell culture.

-   122. The method of Claim 121, wherein the neurological disorder is    an increased anxiety-like response during open field activity    testing.-   123. The method of Claim 121, wherein the neurological disorder is a    decreased anxiety-like response during open field activity testing.-   124. The method of Claim 121, wherein the neurological disorder is    an abnormal circadian rhythm during home-cage activity testing.-   125. The method of Claim 121, wherein the neurological disorder is    an enhanced motor coordination during inverted screen testing.-   126. The method of Claim 121, wherein the neurological disorder is    an impaired motor coordination during inverted screen testing.-   127. The method of Claim 121, wherein the neurological disorder is    depression, generalized anxiety disorders, attention deficit    disorder, sleep disorder, hyperactivity disorder, obsessive    compulsive disorder, schizophrenia, cognitive disorders,    hyperalgesia or sensory disorders.-   128. The method of Claim 121, wherein the eye abnormality is a    retinal abnormality.-   129. The method of Claim 121, wherein the eye abnormality is    consistent with vision problems or blindness.-   130. The method of Claim 128, wherein the retinal abnormality is    consistent with retinitis pigmentosa.-   131. The method of Claim 128, wherein the retinal abnormality is    characterized by retinal degeneration or retinal dysplasia.-   132. The method of Claim 128, wherein the retinal abnormality is    consistent with retinal dysplasia, various retinopathies, including    retinopathy of prematurity, retrolental fibroplasia, neovascular    glaucoma, age-related macular degeneration, diabetic macular edema,    corneal neovascularization, corneal graft neovascularization,    corneal graft rejection, retinal/choroidal neovascularization,    neovascularization of the angle (rubeosis), ocular neovascular    disease, vascular restenosis, arteriovenous malformations (AVM),    meningioma, hemangioma, angiofibroma, thyroid hyperplasias    (including Grave's disease), corneal and other tissue    transplantation, retinal artery obstruction or occlusion; retinal    degeneration causing secondary atrophy of the retinal vasculature,    retinitis pigmentosa, macular dystrophies, Stargardt's disease,    congenital stationary night blindness, choroideremia, gyrate    atrophy, Leber's congenital amaurosis, retinoschisis disorders,    Wagner's syndrome, Usher syndromes, Zellweger syndrome,    Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl    syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's    syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird    syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,    Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,    Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,    olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler    syndrome, carotinemeia, cystinosis, Wolfram syndrome,    Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia    pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,    or mannosidosis.-   133. The method of Claim 121, wherein the eye abnormality is a    cataract.-   134. The method of Claim 133, wherein the cataract is a systemic    disease such as human Down's syndrome, Hallerman-Streiff syndrome,    Lowe syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport    syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or    Conradi syndrome.-   135. The method of Claim 121, wherein the developmental abnormality    comprises embryonic lethality or reduced viability.-   136. The method of Claim 121, wherein the cardiovascular,    endothelial or angiogenic disorders are arterial diseases, such as    diabetes mellitus; papilledema; optic atrophy; atherosclerosis;    angina; myocardial infarctions such as acute myocardial infarctions,    cardiac hypertrophy, and heart failure such as congestive heart    failure; hypertension; inflammatory vasculitides; Reynaud's disease    and Reynaud's phenomenon; aneurysms and arterial restenosis; venous    and lymphatic disorders such as thrombophlebitis, lymphangitis, and    lymphedema; peripheral vascular disease; cancer such as vascular    tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,    telangiectasia, bacillary angiomatosis, hemangioendothelioma,    angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,    and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,    burns, and other injured tissue, implant fixation, scarring;    ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular    disease; renal diseases such as acute renal failure, or    osteoporosis.-   137. The method of Claim 121, wherein the immunological disorders    are systemic lupus erythematosis; rheumatoid arthritis; juvenile    chronic arthritis; spondyloarthropathies; systemic sclerosis    (scleroderma); idiopathic inflammatory myopathies (dermatomyositis,    polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;    autoimmune hemolytic anemia (immune pancytopenia, paroxysmal    nocturnal hemoglobinuria); autoimmune thrombocytopenia (idiopathic    thrombocytopenic purpura, immune-mediated thrombocytopenia);    thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile    lymphocytic thyroiditis, atrophic thyroiditis); diabetes mellitus;    immune-mediated renal disease (glomerulonephritis,    tubulointerstitial nephritis); demyelinating diseases of the central    and peripheral nervous systems such as multiple sclerosis,    idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome,    and chronic inflammatory demyelinating polyneuropathy; hepatobiliary    diseases such as infectious hepatitis (hepatitis A, B, C, D, E and    other non-hepatotropic viruses), autoimmune chronic active    hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and    sclerosing cholangitis; inflammatory bowel disease (ulcerative    colitis: Crohn's disease); gluten-sensitive enteropathy, and    Whipple's disease; autoimmune or immune-mediated skin diseases    including bullous skin diseases, erythema multiforme and contact    dermatitis, psoriasis; allergic diseases such as asthma, allergic    rhinitis, atopic dermatitis, food hypersensitivity and urticaria;    immunologic diseases of the lung such as eosinophilic pneumonia,    idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; or    transplantation associated diseases including graft rejection and    graft-versus-host disease.-   138. The method of Claim 121, wherein said bone metabolic    abnormality or disorder is arthritis, osteoporosis or osteopetrosis.-   139. An agent identified by the method of Claim 121.-   140. The agent of Claim 139 which is an agonist or antagonist of a    PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,    PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,    PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,    PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,    PRO10282, PRO61709 or PRO779 polypeptide.-   141. The agent of Claim 140, wherein the agonist is an anti-PRO196,    anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,    anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,    anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,    anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,    anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,    anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,    anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or    anti-PRO779 antibody.-   142. The agent of Claim 140, wherein the antagonist is an    anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,    anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,    anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,    anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,    anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,    anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,    anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,    anti-PRO61709 or anti-PRO779 antibody.-   143. A therapeutic agent identified by the method of Claim 121.-   144. A method of modulating a phenotype associated with a disruption    of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,    PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,    PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,    PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,    PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779    polypeptide, the method comprising administering to a subject whom    may already have the phenotype, or may be prone to have the    phenotype or may be in whom the phenotype is to be prevented, an    effective amount of the agent of Claim 46, or agonists or    antagonists thereof, thereby effectively modulating the phenotype.-   145. A method of modulating a physiological characteristic    associated with a disruption of a gene which encodes for a PRO196,    PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,    PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,    PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,    PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,    PRO61709 or PRO779 polypeptide, the method comprising administering    to a subject whom may already exhibit the physiological    characteristic, or may be prone to exhibit the physiological    characteristic or may be in whom the physiological characteristic is    to be prevented, an effective amount of the agent of Claim 52, or    agonists or antagonists thereof, thereby effectively modulating the    physiological characteristic.-   146. A method of modulating a behavior associated with a disruption    of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,    PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,    PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,    PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,    PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779    polypeptide, the method comprising administering to a subject whom    may already exhibit the behavior, or may be prone to exhibit the    behavior or may be in whom the exhibited behavior is to be    prevented, an effective amount of the agent of Claim 63, or agonists    or antagonists thereof, thereby effectively modulating the behavior.-   147. A method of modulating the expression of a PRO196, PRO217,    PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,    PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,    PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,    PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or    PRO779 polypeptide, the method comprising administering to a host    cell expressing said PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,    PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,    PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,    PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,    PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, an    effective amount of the agent of claim 92, or agonists or    antagonists thereof, thereby effectively modulating the expression    of said polypeptide.-   148. A method of modulating a condition associated with a disruption    of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,    PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,    PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,    PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,    PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779    polypeptide, the method comprising administering to a subject whom    may have the condition, or may be prone to have the condition or may    be in whom the condition is to be prevented, a therapeutically    effective amount of the therapeutic agent of claim 98, or agonists    or antagonists thereof, thereby effectively modulating the    condition.-   149. A method of treating or preventing or ameliorating a    neurological disorder; cardiovascular, endothelial or angiogenic    disorder; immunological disorder; oncological disorder; bone    metabolic abnormality or disorder, or embryonic lethality associated    with the disruption of a gene which encodes for a PRO196, PRO217,    PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,    PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,    PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,    PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or    PRO779 polypeptide, the method comprising administering to a    non-human transgenic animal cell culture, each cell of said culture    comprising a disruption of the gene which encodes for a PRO196,    PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,    PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,    PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,    PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,    PRO61709 or PRO779 polypeptide, a therapeutically effective amount    of the agent of Claim 139, or agonists or antagonists thereof,    thereby effectively treating or preventing or ameliorating said    disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO196 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA22779-1130” (UNQ170).

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO217 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA33094-1131” (UNQ191).

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequencePRO231 cDNA, wherein SEQ ID NO:5 is a clone designated herein as“DNA34434-1139” (UNQ205).

FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequencePRO236 cDNA, wherein SEQ ID NO:7 is a clone designated herein as“DNA35599-1168” (UNQ210).

FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived from thecoding sequence of SEQ ID NO:7 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native sequencePRO245 cDNA, wherein SEQ ID NO:9 is a clone designated herein as“DNA35638-1141” (UNQ219).

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived from thecoding sequence of SEQ ID NO:9 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO246 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA35639-1172” (UNQ220).

FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequencePRO258 cDNA, wherein SEQ ID NO:13 is a clone designated herein as“DNA35918-1174” (UNQ225).

FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived from thecoding sequence of SEQ ID NO:13 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO287 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA39969-1185” (UNQ250).

FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:15 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO328 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA40587-1231” (UNQ289).

FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 17.

FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequencePRO344 cDNA, wherein SEQ ID NO:19 is a clone designated herein as“DNA40592-1242” (UNQ303).

FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived from thecoding sequence of SEQ ID NO:19 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequencePRO357 cDNA, wherein SEQ ID NO:21 is a clone designated herein as“DNA44804-1248” (UNQ314).

FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:21 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO526 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA44184-1319” (UNQ330).

FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a native sequencePRO724 cDNA, wherein SEQ ID NO:25 is a clone designated herein as“DNA49631-1328” (UNQ389).

FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived from thecoding sequence of SEQ ID NO:25 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO731 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“DNA48331-1329” (UNQ395).

FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a native sequencePRO732 cDNA, wherein SEQ ID NO:29 is a clone designated herein as“DNA48334-1435” (UNQ396).

FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived from thecoding sequence of SEQ ID NO:29 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a native sequencePRO1003 cDNA, wherein SEQ ID NO:31 is a clone designated herein as“DNA58846-1409” (UNQ487).

FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived from thecoding sequence of SEQ ID NO:31 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequencePRO1104 cDNA, wherein SEQ ID NO:33 is a clone designated herein as“DNA59616-1465” (UNQ547).

FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived from thecoding sequence of SEQ ID NO:33 shown in FIG. 33.

FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequencePRO1151 cDNA, wherein SEQ ID NO:35 is a clone designated herein as“DNA44694-1500” (UNQ581).

FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived from thecoding sequence of SEQ ID NO:35 shown in FIG. 35.

FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a native sequencePRO1244 cDNA, wherein SEQ ID NO:37 is a clone designated herein as“DNA64883-1526” (UNQ628).

FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived from thecoding sequence of SEQ ID NO:37 shown in FIG. 37.

FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a native sequencePRO1298 cDNA, wherein SEQ ID NO:39 is a clone designated herein as“DNA66511-1563” (UNQ666).

FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived from thecoding sequence of SEQ ID NO:39 shown in FIG. 39.

FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a native sequencePRO1313 cDNA, wherein SEQ ID NO:41 is a clone designated herein as“DNA64966-1575” (UNQ679).

FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived from thecoding sequence of SEQ ID NO:41 shown in FIG. 41.

FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a native sequencePRO1570 cDNA, wherein SEQ ID NO:43 is a clone designated herein as“DNA68885-1678” (UNQ776).

FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived from thecoding sequence of SEQ ID NO:43 shown in FIG. 43.

FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a native sequencePRO1886 cDNA, wherein SEQ ID NO:45 is a clone designated herein as“DNA80796-2523” (UNQ870).

FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived from thecoding sequence of SEQ ID NO:45 shown in FIG. 45.

FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequencePRO1891 cDNA, wherein SEQ ID NO:47 is a clone designated herein as“DNA76788-2526” (UNQ873).

FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived from thecoding sequence of SEQ ID NO:47 shown in FIG. 47.

FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a native sequencePRO4409 cDNA, wherein SEQ ID NO:49 is a clone designated herein as“DNA88004-2575” (UNQ1934).

FIG. 50 shows the amino acid sequence (SEQ ID NO:50) derived from thecoding sequence of SEQ ID NO:49 shown in FIG. 49.

FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequencePRO5725 cDNA, wherein SEQ ID NO:51 is a clone designated herein as“DNA92265-2669” (UNQ2446).

FIG. 52 shows the amino acid sequence (SEQ ID NO:52) derived from thecoding sequence of SEQ ID NO:51 shown in FIG. 51.

FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a native sequencePRO5994 cDNA, wherein SEQ ID NO:53 is a clone designated herein as“DNA98591” (UNQ2506).

FIG. 54 shows the amino acid sequence (SEQ ID NO:54) derived from thecoding sequence of SEQ ID NO:53 shown in FIG. 53.

FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequencePRO6097 cDNA, wherein SEQ ID NO:55 is a clone designated herein as“DNA107701-2711” (UNQ2545).

FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived from thecoding sequence of SEQ ID NO:55 shown in FIG. 55.

FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequencePRO7425 cDNA, wherein SEQ ID NO:57 is a clone designated herein as“DNA108792-2753” (UNQ2966).

FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from thecoding sequence of SEQ ID NO:57 shown in FIG. 57.

FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequencePRO10102 cDNA, wherein SEQ ID NO:59 is a clone designated herein as“DNA129542-2808” (UNQ3103).

FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from thecoding sequence of SEQ ID NO:59 shown in FIG. 59.

FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a native sequencePRO10282 cDNA, wherein SEQ ID NO:61 is a clone designated herein as“DNA148380-2827” (UNQ3126).

FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from thecoding sequence of SEQ ID NO:61 shown in FIG. 61.

FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO61709 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“DNA347767” (UNQ14964).

FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 63.

FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a native sequencePRO779 cDNA, wherein SEQ ID NO:65 is a clone designated herein as“DNA58801-1052” (UNQ455).

FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from thecoding sequence of SEQ ID NO:65 shown in FIG. 65.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides described herein may beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.The term “PRO polypeptide” refers to each individual PRO/numberpolypeptide disclosed herein. All disclosures in this specificationwhich refer to the “PRO polypeptide” refer to each of the polypeptidesindividually as well as jointly. For example, descriptions of thepreparation of, purification of, derivation of, formation of antibodiesto or against, administration of, compositions containing, treatment ofa disease with, etc., pertain to each polypeptide of the inventionindividually. The term “PRO polypeptide” also includes variants of thePRO/number polypeptides disclosed herein.

A “native sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide” comprises a polypeptide havingthe same amino acid sequence as the corresponding PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidederived from nature. Such native sequence PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. Theinvention provides native sequence PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides disclosed hereinwhich are mature or full-length native sequence polypeptides comprisingthe full-length amino acids sequences shown in the accompanying figures.Start and stop codons are shown in bold font and underlined in thefigures. However, while the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide disclosed in the accompanyingfigures are shown to begin with methionine residues designated herein asamino acid position 1 in the figures, it is conceivable and possiblethat other methionine residues located either upstream or downstreamfrom the amino acid position 1 in the figures may be employed as thestarting amino acid residue for the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides.

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide “extracellular domain” or “ECD” refers toa form of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are contemplated by thepresent invention.

The approximate location of the “signal peptides” of the various PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide variant” means a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, preferably an activePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide, as defined herein having at least about 80% aminoacid sequence identity with a full-length native sequence PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide sequence as disclosed herein, a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, with or without the signal peptide, asdisclosed herein or any other fragment of a full-length PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesequence as disclosed herein (such as those encoded by a nucleic acidthat represents only a portion of the complete coding sequence for afull-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide). Such PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide variants include, forinstance, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the full-length nativeamino acid sequence. Ordinarily, a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide variant will have orwill have at least about 80% amino acid sequence identity, alternativelywill have or will have at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity, to a full-length native sequence PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesequence as disclosed herein, a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, with or without the signal peptide, as disclosed herein orany other specifically defined fragment of a full-length PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesequence as disclosed herein. Ordinarily, PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variant polypeptides areor are at least about 10 amino acids in length, alternatively are or areat least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600 amino acids in length, or more. Optionally, PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779variant polypeptides will have no more than one conservative amino acidsubstitution as compared to the native PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence,alternatively will have or will have no more than 2, 3, 4, 5, 6, 7, 8,9, or 10 conservative amino acid substitution as compared to the nativePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide sequence.

“Percent (%) amino acid sequence identity” with respect to the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues. Unlessspecifically stated otherwise, all % amino acid sequence identity valuesused herein are obtained as described in the immediately precedingparagraph using the ALIGN-2 computer program.

“PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 variant polynucleotide” or “PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 variant nucleic acid sequence”means a nucleic acid molecule which encodes a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, preferablyan active PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, as defined herein and which has at leastabout 80% nucleic acid sequence identity with a nucleotide acid sequenceencoding a full-length native sequence PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence as disclosedherein, a full-length native sequence PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence lacking thesignal peptide as disclosed herein, an extracellular domain of a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence as disclosedherein (such as those encoded by a nucleic acid that represents only aportion of the complete coding sequence for a full-length PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide). Ordinarily, a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 variant polynucleotide will have or willhave at least about 80% nucleic acid sequence identity, alternativelywill have or will have at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleicacid sequence identity with a nucleic acid sequence encoding afull-length native sequence PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide sequence as disclosed herein, afull-length native sequence PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence as disclosedherein. Variants do not encompass the native nucleotide sequence.

Ordinarily, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 variant polynucleotides are or are at least about 5nucleotides in length, alternatively are or are at least about 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700,710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, or 1000 nucleotides in length, wherein in this context the term“about” means the referenced nucleotide sequence length plus or minus10% of that referenced length.

“Percent (%) nucleic acid sequence identity” with respect to PRO196-,PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-,PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-encoding nucleic acid sequencesidentified herein is defined as the percentage of nucleotides in acandidate sequence that are identical with the nucleotides in thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 nucleic acid sequence of interest, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percent nucleicacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.For purposes herein, however, % nucleic acid sequence identity valuesare generated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 1 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 1 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides. Unless specifically statedotherwise, all % nucleic acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The invention also provides PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 variant polynucleotides which are nucleicacid molecules that encode a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide as disclosed herein. PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 variant polypeptides may be those that areencoded by a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 variant polynucleotide.

The term “full-length coding region” when used in reference to a nucleicacid encoding a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide refers to the sequence of nucleotideswhich encode the full-length PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide of the invention (which isoften shown between start and stop codons, inclusive thereof, in theaccompanying figures). The term “full-length coding region” when used inreference to an ATCC deposited nucleic acid refers to the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide-encoding portion of the cDNA that is inserted into thevector deposited with the ATCC (which is often shown between start andstop codons, inclusive thereof, in the accompanying figures).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. The invention provides that the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

“Active” or “activity” for the purposes herein refers to form(s) of aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, wherein“biological” activity refers to a biological function (either inhibitoryor stimulatory) caused by a native or naturally-occurring PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide other than the ability to induce the production of anantibody against an antigenic epitope possessed by a native ornaturally-occurring PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide and an “immunological” activityrefers to the ability to induce the production of an antibody against anantigenic epitope possessed by a native or naturally-occurring PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide.

The term “antagonist” is used in the broadest sense [unless otherwisequalified], and includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of a native PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide disclosed herein. In a similar manner, the term “agonist” isused in the broadest sense [unless otherwise qualified] and includes anymolecule that mimics a biological activity of a native PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidedisclosed herein. Suitable agonist or antagonist molecules specificallyinclude agonist or antagonist antibodies or antibody fragments,fragments or amino acid sequence variants of native PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides,peptides, antisense oligonucleotides, small organic molecules, etc.Methods for identifying agonists or antagonists of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide maycomprise contacting a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide with a candidate agonist orantagonist molecule and measuring a detectable change in one or morebiological activities normally associated with the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. A subject in need of treatment may already have the disorder,or may be prone to have the disorder or may be in whom the disorder isto be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents such as rats or mice, domestic andfarm animals, and zoo, sports, or pet animals, such as dogs, cats,cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammalis human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Dependingon the context, the solid phase can comprise the well of an assay plate;in others it is a purification column (e.g., an affinity chromatographycolumn). This term also includes a discontinuous solid phase of discreteparticles, such as those described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

An “effective amount” of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody, a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 binding oligopeptide, a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding organicmolecule or an agonist or antagonist thereof as disclosed herein is anamount sufficient to carry out a specifically stated purpose. An“effective amount” may be determined empirically and in a routinemanner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of ananti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 binding oligopeptide, a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 binding organic molecule or other drugeffective to “treat” a disease or disorder in a subject or mammal. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. See the definition herein of “treating”. To the extent the drugmay prevent growth and/or kill existing cancer cells, it may becytostatic and/or cytotoxic.

The phrases “cardiovascular, endothelial and angiogenic disorder”,“cardiovascular, endothelial and angiogenic dysfunction”,“cardiovascular, endothelial or angiogenic disorder” and“cardiovascular, endothelial or angiogenic dysfunction” are usedinterchangeably and refer in part to systemic disorders that affectvessels, such as diabetes mellitus, as well as diseases of the vesselsthemselves, such as of the arteries, capillaries, veins, and/orlymphatics. This would include indications that stimulate angiogenesisand/or cardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatoryvasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; and other vasculardisorders such as peripheral vascular disease, cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, or osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as CHF.

“Hypertrophy”, as used herein, is defined as an increase in mass of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both. Certain organs, such as the heart, lose the ability to divideshortly after birth. Accordingly, “cardiac hypertrophy” is defined as anincrease in mass of the heart, which, in adults, is characterized by anincrease in myocyte cell size and contractile protein content withoutconcomitant cell division. The character of the stress responsible forinciting the hypertrophy, (e.g., increased preload, increased afterload,loss of myocytes, as in myocardial infarction, or primary depression ofcontractility), appears to play a critical role in determining thenature of the response. The early stage of cardiac hypertrophy isusually characterized morphologically by increases in the size ofmyofibrils and mitochondria, as well as by enlargement of mitochondriaand nuclei. At this stage, while muscle cells are larger than normal,cellular organization is largely preserved. At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size ornumber of specific organelles, such as mitochondria, and new contractileelements are added in localized areas of the cells, in an irregularmanner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nucleiwith highly lobulated membranes, which displace adjacent myofibrils andcause breakdown of normal Z-band registration. The phrase “cardiachypertrophy” is used to include all stages of the progression of thiscondition, characterized by various degrees of structural damage of theheart muscle, regardless of the underlying cardiac disorder. Hence, theterm also includes physiological conditions instrumental in thedevelopment of cardiac hypertrophy, such as elevated blood pressure,aortic stenosis, or myocardial infarction.

“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. The heart failure can be caused by a number offactors, including ischemic, congenital, rheumatic, or idiopathic forms.

“Congestive heart failure” (CHF) is a progressive pathologic state wherethe heart is increasingly unable to supply adequate cardiac output (thevolume of blood pumped by the heart over time) to deliver the oxygenatedblood to peripheral tissues. As CHF progresses, structural andhemodynamic damages occur. While these damages have a variety ofmanifestations, one characteristic symptom is ventricular hypertrophy.CHF is a common end result of a number of various cardiac disorders.

“Myocardial infarction” generally results from atherosclerosis of thecoronary arteries, often with superimposed coronary thrombosis. It maybe divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

As a result of the increased stress or strain placed on the heart inprolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506(1984); Smith et al., J. Am. Coll. Cardiol., 5: 869-874 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supranormal systolicfunction. Hartford et al., Hypertension, 6: 329-338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular hypertrophy, without improvement indiastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7 (1984).

Another complex cardiac disease associated with cardiac hypertrophy is“hypertrophic cardiomyopathy”. This condition is characterized by agreat diversity of morphologic, functional, and clinical features (Maronet al., N. Engl. J. Med., 316: 780-789 (1987); Spirito et al., N. Engl.J. Med., 320: 749-755 (1989); Louie and Edwards, Prog. Cardiovasc. Dis.,36: 275-308 (1994); Wigle et al., Circulation, 92: 1680-1692 (1995)),the heterogeneity of which is accentuated by the fact that it afflictspatients of all ages. Spirito et al., N. Engl. J. Med., 336: 775-785(1997). The causative factors of hypertrophic cardiomyopathy are alsodiverse and little understood. In general, mutations in genes encodingsarcomeric proteins are associated with hypertrophic cardiomyopathy.Recent data suggest that β-myosin heavy chain mutations may account forapproximately 30 to 40 percent of cases of familial hypertrophiccardiomyopathy. Watkins et al., N. Engl. J. Med., 326: 1108-1114 (1992);Schwartz et al, Circulation, 91: 532-540 (1995); Marian and Roberts,Circulation, 92: 1336-1347 (1995); Thierfelder et al., Cell, 77: 701-712(1994); Watkins et al., Nat. Gen., 11: 434-437 (1995). Besides β-myosinheavy chain, other locations of genetic mutations include cardiactroponin T, alpha topomyosin, cardiac myosin binding protein C,essential myosin light chain, and regulatory myosin light chain. See,Malik and Watkins, Curr. Opin. Cardiol., 12: 295-302 (1997).

Supravalvular “aortic stenosis” is an inherited vascular disordercharacterized by narrowing of the ascending aorta, but other arteries,including the pulmonary arteries, may also be affected. Untreated aorticstenosis may lead to increased intracardiac pressure resulting inmyocardial hypertrophy and eventually heart failure and death. Thepathogenesis of this disorder is not fully understood, but hypertrophyand possibly hyperplasia of medial smooth muscle are prominent featuresof this disorder. It has been reported that molecular variants of theelastin gene are involved in the development and pathogenesis of aorticstenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.

“Valvular regurgitation” occurs as a result of heart diseases resultingin disorders of the cardiac valves. Various diseases, like rheumaticfever, can cause the shrinking or pulling apart of the valve orifice,while other diseases may result in endocarditis, an inflammation of theendocardium or lining membrane of the atrioventricular orifices andoperation of the heart. Defects such as the narrowing of the valvestenosis or the defective closing of the valve result in an accumulationof blood in the heart cavity or regurgitation of blood past the valve.If uncorrected, prolonged valvular stenosis or insufficiency may resultin cardiac hypertrophy and associated damage to the heart muscle, whichmay eventually necessitate valve replacement.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, include systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, or transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includingviral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, andE, herpes, etc., bacterial infections, fungal infections, protozoalinfections and parasitic infections.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis, and ankylosing spondylitis),psoriasis, dermatitis including atopic dermatitis; chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, toxic epidermal necrolysis, systemicscleroderma and sclerosis, responses associated with inflammatory boweldisease (IBD) (Crohn's disease, ulcerative colitis), and IBD withco-segregate of pyoderma gangrenosum, erythema nodosum, primarysclerosing cholangitis, and/or episcleritis), respiratory distresssyndrome, including adult respiratory distress syndrome (ARDS),meningitis, IgE-mediated diseases such as anaphylaxis and allergicrhinitis, encephalitis such as Rasmussen's encephalitis, uveitis,colitis such as microscopic colitis and collagenous colitis,glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN,membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, eczema, asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency,systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus(including nephritis, cerebritis, pediatric, non-renal, discoid,alopecia), juvenile onset diabetes, multiple sclerosis (MS) such asspino-optical MS, allergic encephalomyelitis, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includingWegener's granulomatosis, agranulocytosis, vasculitis (including LargeVessel vasculitis (including Polymyalgia Rheumatica and Giant Cell(Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki'sDisease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associatedvasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency,hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseasesinvolving leukocyte diapedesis, CNS inflammatory disorders, multipleorgan injury syndrome, myasthenia gravis, antigen-antibody complexmediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease,Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton MyasthenicSyndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnsonsyndrome, solid organ transplant rejection (including pretreatment forhigh panel reactive antibody titers, IgA deposit in tissues, andrejection arising from renal transplantation, liver transplantation,intestinal transplantation, cardiac transplantation, etc.), graft versushost disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris,foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, immunecomplex nephritis, IgM polyneuropathies or IgM mediated neuropathy,idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenicpurpura (TTP), thrombocytopenia (as developed by myocardial infarctionpatients, for example), including autoimmune thrombocytopenia,autoimmune disease of the testis and ovary including autoimune orchitisand oophoritis, primary hypothyroidism; autoimmune endocrine diseasesincluding autoimmune thyroiditis, chronic thyroiditis (Hashimoto'sThyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison'sdisease, Grave's disease, autoimmune polyglandular syndromes (orpolyglandular endocrinopathy syndromes), Type I diabetes also referredto as insulin-dependent diabetes mellitus (IDDM), including pediatricIDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré Syndrome, Berger's Disease (IgAnephropathy), primary biliary cirrhosis, celiac sprue (glutenenteropathy), refractory sprue with co-segregate dermatitisherpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune inner eardisease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome(OMS), polychondritis such as refractory polychondritis, pulmonaryalveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,monoclonal gammopathy of uncertain/unknown significance (MGUS),peripheral neuropathy, paraneoplastic syndrome, channelopathies such asepilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,periodic paralysis, and channelopathies of the CNS; autism, inflammatorymyopathy, and focal segmental glomerulosclerosis (FSGS).

The phrase “anxiety related disorders” refers to disorders of anxiety,mood, and substance abuse, including but not limited to: depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Such disorders include the mild to moderate anxiety, anxiety disorderdue to a general medical condition, anxiety disorder not otherwisespecified, generalized anxiety disorder, panic attack, panic disorderwith agoraphobia, panic disorder without agoraphobia, posttraumaticstress disorder, social phobia, social anxiety, autism, specific phobia,substance-induced anxiety disorder, acute alcohol withdrawal, obsessivecompulsive disorder, agoraphobia, monopolar disorders, bipolar disorderI or II, bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder, enhancement of cognitive function, lossof cognitive function associated with but not limited to Alzheimer'sdisease, stroke, or traumatic injury to the brain, seizures resultingfrom disease or injury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

The term “lipid metabolic disorder” refers to abnormal clinicalchemistry levels of cholesterol and triglycerides, wherein elevatedlevels of these lipids is an indication for atherosclerosis.Additionally, abnormal serum lipid levels may be an indication ofvarious cardiovascular diseases including hypertension, stroke, coronaryartery diseases, diabetes and/or obesity.

The phrase “eye abnormality” refers to such potential disorders of theeye as they may be related to atherosclerosis or variousophthalmological abnormalities. Such disorders include but are notlimited to the following: retinal dysplasia, various retinopathies,restenosis, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis. Cataracts are also considered an eye abnormality and areassociated with such systemic diseases as: Human Down's syndrome,Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfansyndrome, Trismoy 13-15 condition, Alport syndrome, myotonic dystrophy,Fabry disease, hypothroidisms, or Conradi syndrome. Other oculardevelopmental anomalies include: Aniridia, anterior segment anddysgenesis syndrome. Cataracts may also occur as a result of anintraocular infection or inflammation (uveitis).

A “growth inhibitory amount” of an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding oligopeptide orPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 binding organic molecule is an amount capable of inhibiting thegrowth of a cell, especially tumor, e.g., cancer cell, either in vitroor in vivo. A “growth inhibitory amount” of an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding oligopeptide orPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 binding organic molecule for purposes of inhibiting neoplasticcell growth may be determined empirically and in a routine manner.

A “cytotoxic amount” of an anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 binding oligopeptide or PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding organicmolecule is an amount capable of causing the destruction of a cell,especially tumor, e.g., cancer cell, either in vitro or in vivo. A“cytotoxic amount” of an anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 binding oligopeptide or PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding organicmolecule for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody monoclonal antibodies (including agonist, antagonist, andneutralizing antibodies), anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody compositions with polyepitopic specificity, polyclonalantibodies, single chain anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibodies, and fragments of anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibodies (see below) as long as they exhibit the desired biological orimmunological activity. The term “immunoglobulin” (Ig) is usedinterchangeable with antibody herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. The invention provides that the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the V_(H); Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2 and C_(H)3. The constant domains may be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody,” e.g., a mammalian anti-human IgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

A “PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 binding oligopeptide” is an oligopeptide that binds,preferably specifically, to a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide as described herein. PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779binding oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 binding oligopeptides usually are or are atleast about 5 amino acids in length, alternatively are or are at leastabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein sucholigopeptides that are capable of binding, preferably specifically, to aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide as described herein. PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 binding oligopeptides may beidentified without undue experimentation using well known techniques. Inthis regard, it is noted that techniques for screening oligopeptidelibraries for oligopeptides that are capable of specifically binding toa polypeptide target are well known in the art (see, e.g., U.S. Pat.Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564;Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysenet al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen etal., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci.USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991),J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

A “PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 binding organic molecule” is an organic moleculeother than an oligopeptide or antibody as defined herein that binds,preferably specifically, to a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide as described herein. PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779binding organic molecules may be identified and chemically synthesizedusing known methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 binding organic molecules are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic molecules that are capableof binding, preferably specifically, to a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide as describedherein may be identified without undue experimentation using well knowntechniques. In this regard, it is noted that techniques for screeningorganic molecule libraries for molecules that are capable of binding toa polypeptide target are well known in the art (see, e.g., PCTPublication Nos. WO00/00823 and WO00/39585).

An antibody, oligopeptide or other organic molecule “which binds” anantigen of interest, e.g. a tumor-associated polypeptide antigen target,is one that binds the antigen with sufficient affinity such that theantibody, oligopeptide or other organic molecule is preferably useful asa diagnostic and/or therapeutic agent in targeting a cell or tissueexpressing the antigen, and does not significantly cross-react withother proteins. The extent of binding of the antibody, oligopeptide orother organic molecule to a “non-target” protein will be less than about10% of the binding of the antibody, oligopeptide or other organicmolecule to its particular target protein as determined by fluorescenceactivated cell sorting (FACS) analysis or radioimmunoprecipitation(RIA). With regard to the binding of an antibody, oligopeptide or otherorganic molecule to a target molecule, the term “specific binding” or“specifically binds to” or is “specific for” a particular polypeptide oran epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target, for example, an excess ofnon-labeled target. In this case, specific binding is indicated if thebinding of the labeled target to a probe is competitively inhibited byexcess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about 10⁻⁴M, alternatively at least about 10⁻⁵ M, alternatively at least about10⁻⁶ M, alternatively at least about 10⁻⁷ M, alternatively at leastabout 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively atleast about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternativelyat least about 10⁻¹² M, or greater. The term “specific binding” refersto binding where a molecule binds to a particular polypeptide or epitopeon a particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

An antibody, oligopeptide or other organic molecule that “inhibits thegrowth of tumor cells expressing a “PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779” or a “growth inhibitory”antibody, oligopeptide or other organic molecule is one which results inmeasurable growth inhibition of cancer cells expressing oroverexpressing the appropriate PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. The PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide may be atransmembrane polypeptide expressed on the surface of a cancer cell ormay be a polypeptide that is produced and secreted by a cancer cell.Preferred growth inhibitory anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibodies, oligopeptides or organic molecules inhibit growth ofPRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-,PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-expressing tumor cells by or by greaterthan 20%, preferably from about 20% to about 50%, and even morepreferably, by or by greater than 50% (e.g., from about 50% to about100%) as compared to the appropriate control, the control typicallybeing tumor cells not treated with the antibody, oligopeptide or otherorganic molecule being tested. Growth inhibition can be measured at anantibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200nM in cell culture, where the growth inhibition is determined 1-10 daysafter exposure of the tumor cells to the antibody. Growth inhibition oftumor cells in vivo can be determined in various ways. The antibody isgrowth inhibitory in vivo if administration of the anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody at about 1 μg/kg to about 100mg/kg body weight results in reduction in tumor size or tumor cellproliferation within about 5 days to 3 months from the firstadministration of the antibody, preferably within about 5 to 30 days.

An antibody, oligopeptide or other organic molecule which “inducesapoptosis” is one which induces programmed cell death as determined bybinding of annexin V, fragmentation of DNA, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is usually one whichoverexpresses a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. Preferably the cell is a tumor cell,e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney,colon, bladder cell. Various methods are available for evaluating thecellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, theantibody, oligopeptide or other organic molecule which induces apoptosisis one which results in or in about 2 to 50 fold, preferably in or inabout 5 to 50 fold, and most preferably in or in about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci.U.S.A. 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD).Preferably, the cancer comprises a tumor that expresses an IGF receptor,more preferably breast cancer, lung cancer, colorectal cancer, orprostate cancer, and most preferably breast or prostate cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as dodronate; an esperamicin; as wellas neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), adacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one aspect of the invention, the cell proliferativedisorder is cancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

An antibody, oligopeptide or other organic molecule which “induces celldeath” is one which causes a viable cell to become nonviable. The cellis one which expresses a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, preferably a cell thatoverexpresses a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide as compared to a normal cell of the sametissue type. The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide may be a transmembrane polypeptideexpressed on the surface of a cancer cell or may be a polypeptide thatis produced and secreted by a cancer cell. Preferably, the cell is acancer cell, e.g., a breast, ovarian, stomach, endometrial, salivarygland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Celldeath in vitro may be determined in the absence of complement and immuneeffector cells to distinguish cell death induced by antibody-dependentcell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity(CDC). Thus, the assay for cell death may be performed using heatinactivated serum (i.e., in the absence of complement) and in theabsence of immune effector cells. To determine whether the antibody,oligopeptide or other organic molecule is able to induce cell death,loss of membrane integrity as evaluated by uptake of propidium iodide(PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or7AAD can be assessed relative to untreated cells. Preferred celldeath-inducing antibodies, oligopeptides or other organic molecules arethose which induce PI uptake in the PI uptake assay in BT474 cells.

As used herein, the term “immunoadhesion” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesion”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesions comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesion part of an immunoadhesion molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesion may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

“Replication-preventing agent” is an agent wherein replication,function, and/or growth of the cells is inhibited or prevented, or cellsare destroyed, no matter what the mechanism, such as by apoptosis,angiostasis, cytosis, tumoricide, mytosis inhibition, blocking cellcycle progression, arresting cell growth, binding to tumors, acting ascellular mediators, etc. Such agents include a chemotherapeutic agent,cytotoxic agent, cytokine, growth-inhibitory agent, or anti-hormonalagent, e.g., an anti-estrogen compound such as tamoxifen, ananti-progesterone such as onapristone (see, EP 616 812); or ananti-androgen such as flutamide, as well as aromidase inhibitors, or ahormonal agent such as an androgen.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

Preferred cytotoxic agents herein for the specific tumor types to use incombination with the antagonists herein are as follows:

1. Prostate cancer: androgens, docetaxel, paclitaxel, estramustine,doxorubicin, mitoxantrone, antibodies to ErbB2 domain(s) such as 2C4 (WO01/00245; hybridoma ATCC HB-12697), which binds to a region in theextracellular domain of ErbB2 (e.g., any one or more residues in theregion from about residue 22 to about residue 584 of ErbB2, inclusive),AVASTIN™ anti-vascular endothelial growth factor (VEGF), TARCEVA™OSI-774 (erlotinib) (Genenetech and OSI Pharmaceuticals), or otherepidermal growth factor receptor tyrosine kinase inhibitors (EGFRTKI's).

2. Stomach cancer: 5-fluorouracil (5FU), XELODA™ capecitabine,methotrexate, etoposide, cisplatin/carboplatin, pacliitaxel, docetaxel,gemcitabine, doxorubicin, and CPT-11 (camptothcin-11; irinotecan, USABrand Name: CAMPTOSAR®).

3. Pancreatic cancer: gemcitabine, 5FU, XELODA™ capecitabine, CPT-11,docetaxel, paclitaxel, cisplatin, carboplatin, TARCEVA™ erlotinib, andother EGFR TKI's.

4. Colorectal cancer: 5FU, XELODA™ capecitabine, CPT-11, oxaliplatin,AVASTIN™ anti-VEGF, TARCEVA™ erlotinib and other EGFR TKI's, andERBITUX™ (formerly known as IMC-C225) human:murine-chimerized monoclonalantibody that binds to EGFR and blocks the ability of EGF to initiatereceptor activation and signaling to the tumor.

5. Renal cancer: IL-2, interferon alpha, AVASTIN™ anti-VEGF, MEGACE™(Megestrol acetate) progestin, vinblastine, TARCEVA™ erlotinib, andother EGFR TKI's.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a PRO196-,PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-,PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-expressing cancer cell, either in vitroor in vivo. Thus, the growth inhibitory agent may be one whichsignificantly reduces the percentage of PRO196-, PRO217-, PRO231-,PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-,PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-,PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709-or PRO779-expressing cells in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “gene” refers to (a) a gene containing at least one of the DNAsequences disclosed herein; (b) any DNA sequence that encodes the aminoacid sequence encoded by the DNA sequences disclosed herein and/or;{circle around (c)}) any DNA sequence that hybridizes to the complementof the coding sequences disclosed herein. Preferably, the term includescoding as well as noncoding regions, and preferably includes allsequences necessary for normal gene expression.

The term “gene targeting” refers to a type of homologous recombinationthat occurs when a fragment of genomic DNA is introduced into amammalian cell and that fragment locates and recombines with endogenoushomologous sequences. Gene targeting by homologous recombination employsrecombinant DNA technologies to replace specific genomic sequences withexogenous DNA of particular design.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or chromatids at the site ofhomologous nucleotide sequences.

The term “target gene” (alternatively referred to as “target genesequence” or “target DNA sequence”) refers to any nucleic acid molecule,polynucleotide, or gene to be modified by homologous recombination. Thetarget sequence includes an intact gene, an exon or intron, a regulatorysequence or any region between genes. The target gene my comprise aportion of a particular gene or genetic locus in the individual'sgenomic DNA.

“Disruption” of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene occurs when a fragment of genomic DNAlocates and recombines with an endogenous homologous sequence whereinthe disruption is a deletion of the native gene or a portion thereof, ora mutation in the native gene or wherein the disruption is thefunctional inactivation of the native gene. Alternatively, sequencedisruptions may be generated by nonspecific insertional inactivationusing a gene trap vector (i.e. non-human transgenic animals containingand expressing a randomly inserted transgene; see for example U.S. Pat.No. 6,436,707 issued Aug. 20, 2002). These sequence disruptions ormodifications may include insertions, missense, frameshift, deletion, orsubstitutions, or replacements of DNA sequence, or any combinationthereof. Insertions include the insertion of entire genes, which may beof animal, plant, fungal, insect, prokaryotic, or viral origin.Disruption, for example, can alter the normal gene product by inhibitingits production partially or completely or by enhancing the normal geneproduct's activity. Preferably, the disruption is a null disruption,wherein there is no significant expression of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene.

The term “native expression” refers to the expression of the full-lengthpolypeptide encoded by the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene, at expression levels present in thewild-type mouse. Thus, a disruption in which there is “no nativeexpression” of the endogenous PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene refers to a partial or completereduction of the expression of at least a portion of a polypeptideencoded by an endogenous PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene of a single cell, selected cells, orall of the cells of a mammal.

The term “knockout” refers to the disruption of a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene whereinthe disruption results in: the functional inactivation of the nativegene; the deletion of the native gene or a portion thereof; or amutation in the native gene.

The term “knock-in” refers to the replacement of the mouse ortholog (orother mouse gene) with a human cDNA encoding any of the specific humanPRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-,PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-encoding genes or variants thereof (ie.the disruption results in a replacement of a native mouse gene with anative human gene).

The term “construct” or “targeting construct” refers to an artificiallyassembled DNA segment to be transferred into a target tissue, cell lineor animal. Typically, the targeting construct will include a gene or anucleic acid sequence of particular interest, a marker gene andappropriate control sequences. As provided herein, the targetingconstruct comprises a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 targeting construct. A “PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 targetingconstruct” includes a DNA sequence homologous to at least one portion ofa PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 gene and is capable of producing a disruption in aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 gene in a host cell.

The term “transgenic cell” refers to a cell containing within its genomea PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 gene that has been disrupted, modified, altered, orreplaced completely or partially by the method of gene targeting.

The term “transgenic animal” refers to an animal that contains withinits genome a specific gene that has been disrupted or otherwise modifiedor mutated by the methods described herein or methods otherwise wellknown in the art. Preferably the non-human transgenic animal is amammal. More preferably, the mammal is a rodent such as a rat or mouse.In addition, a “transgenic animal” may be a heterozygous animal (i.e.,one defective allele and one wild-type allele) or a homozygous animal(i.e., two defective alleles). An embryo is considered to fall withinthe definition of an animal. The provision of an animal includes theprovision of an embryo or foetus in utero, whether by mating orotherwise, and whether or not the embryo goes to term.

As used herein, the terms “selective marker” and position selectionmarker” refer to a gene encoding a product that enables only the cellsthat carry the gene to survive and/or grow under certain conditions. Forexample, plant and animal cells that express the introduced neomycinresistance (Ned) gene are resistant to the compound G418. Cells that donot carry the Ned gene marker are killed by G418. Other positiveselection markers are known to, or are within the purview of, those ofordinary skill in the art.

The term “modulates” or “modulation” as used herein refers to thedecrease, inhibition, reduction, amelioration, increase or enhancementof a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 gene function, expression, activity, or alternativelya phenotype associated with PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene.

The term “ameliorates” or “amelioration” as used herein refers to adecrease, reduction or elimination of a condition, disease, disorder, orphenotype, including an abnormality or symptom.

The term “abnormality” refers to any disease, disorder, condition, orphenotype in which PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 is implicated, including pathologicalconditions and behavioral observations.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1, 1,0, 0,−6, 0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */{−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */ { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */{−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */ {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */{−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */{−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */ {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */ { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */ { 0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };/*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* maxjumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gapslarger than this */ #define JMPS 1024 /* max jmps in an path */ #defineMX 4 /* save if there's at least MX-1 bases since last jmp */ #defineDMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty formismatched bases */ #define DINS0 8 /* penalty for a gap */ #defineDINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP];/* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no.of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 −1 */struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS];/* size of jmp (gap) */ int x[JMPS];/* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for errmsgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag:nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  *  where file1 and file2 are two dna or twoprotein sequences.  *  The sequences can be in upper- or lower-case anmay contain ambiguity  *  Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  *  Max file length is 65535 (limited by unsigned short x in thejmp struct)  *  A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  *  Output is in the file “align.out”  *  * Theprogram may create a tmp file in /tmp to hold info about traceback.  *Original version developed under BSD 4.3 on a vax 8650  */ #include“nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’-‘A’))|(1<<(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’-‘A’))|(1<<(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\”align.out\“\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */} /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row( ), row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy); if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx< len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax= col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void)free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print( ) -- only routinevisible outside this module  *  * static:  * getmat( ) -- trace backbest path, count matches: print( )  * pr_align( ) -- print alignment ofdescribed in array p[ ]: print( )  * dumpblock( ) -- dump a block oflines with numbers, stars: pr_align( )  * nums( ) -- put out a numberline: dumpblock( )  * putline( ) -- put out a line (name, [num], seq,[num]): dumpblock( )  * stars( ) - -put a line of stars: dumpblock( )  *stripname( ) -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC 3 #define P_LINE 256 /* maximum output line */#define P_SPC 3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print( ) print { int lx, ly, firstgap, lastgap; /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr,“%s: can't write %s\n”,prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s (length =%d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s (length =%d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1; firstgap =lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */ pp[0].spc =firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if (dmax > len1 − 1){ /* leading gap in y */ pp[1].spc = firstgap = dmax − (len1 − 1); lx −=pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gap in x */ lastgap =len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 > len0 − 1) { /*trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −= lastgap; }getmat(lx, ly, firstgap, lastgap); pr_align( ); } /*  * trace back thebest path, count matches  */ static getmat(lx, ly, firstgap, lastgap)getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap, lastgap;/* leading trailing overlap */ { int nm, i0, i1, siz0, siz1; charoutx[32]; double pct; register n0, n1; register char *p0, *p1; /* gettotal matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “”:“s”); fprintf(fx,“%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy ==1)? “”:“s”); fprintf(fx,“%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); }  static nm; /*matches in core -- for checking */  static lmax; /* lengths of strippedfile names */  static ij[2]; /* jmp index for a path */  static nc[2];/* number at start of current line */  static ni[2]; /* current elemnumber -- for gapping */  static siz[2];  static char *ps[2]; /* ptr tocurrent element */  static char *po[2]; /* ptr to next output char slot*/  static char out[2][P_LINE]; /* output line */  static charstar[P_LINE]; /* set by stars( ) */ /*  * print alignment of describedin struct path pp[ ]  */ static pr_align( ) pr_align { int nn; /* charcount */ int more; register I; for (I = 0, lmax = 0; I < 2; I++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more; ) { ...pr_align for (I = more = 0; I < 2; I++) { /*  *do we have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) { /* leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if(siz[i]) { /* in a gap */ *po[i]++ = ‘-’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock( ); for (I = 0; I < 2; I++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register I; for(I = 0; I < 2; I++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (I = 0; I < 2; I++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) !=‘ ’)) { if (I == 0) nums(I); if (I == 0 && *out[1]) stars( );putline(I); if (I == 0 && *out[1]) fprintf(fx, star); if (I == 1)nums(I); } } } /*  * put out a number line: dumpblock( )  */ staticnums(ix) nums int ix; /* index in out[ ] holding seq line */ { charnline[P_LINE]; register I, j; register char *pn, *px, *py; for (pn =nline, I = 0; I < lmax+P_SPC; I++, pn++) *pn = ‘ ’; for (I = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py == ‘ ’ || *py == ‘-’) *pn = ‘ ’;else { if (I%10 == 0 || (I == 1 && nc[ix] != 1)) { j = (I < 0)? −I : I;for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (I < 0) *px = ‘-’;} else *pn = ‘ ’; I++; } } *pn = ‘\0’; nc[ix] = I; for (pn = nline; *pn;pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out aline (name, [num], seq, [num]): dumpblock( )  */ static putline(ix)putline int ix; { ...putline int I; register char *px; for (px =namex[ix], I = 0; *px && *px != ‘:’; px++, I++) (void) putc(*px, fx);for (; I < lmax+P_SPC; I++) (void) putc(‘ ’, fx); /* these count from 1: * ni[ ] is current element (from 1)  * nc[ ] is number at start ofcurrent line  */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx); (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock( )  */ static stars( ) stars { int I;register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ‘ ’ &&*(po[0]) == ‘ ’) ||   !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’))return; px = star; for (I = lmax+P_SPC; I; I−−) *px++ = ‘ ’; for (p0 =out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(I) cleanup int I; { if (fj)(void) unlink(jname); exit(I); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,“r”)) == 0) {fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s: malloc( ) failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register I, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (I = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; I++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (I >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { I = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = I; I = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = I; } for (j = 0, i1−−; j < i1; j++, i1−−) { I= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = I; I = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = I; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw( )  */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%

II. Compositions and Methods of the Invention

A. Full-Length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides. In particular, cDNAs encoding variousPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides have been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

B. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 Polypeptide Variants

In addition to the full-length native sequence PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides describedherein, it is contemplated that PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 variants can be prepared. PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variants can beprepared by introducing appropriate nucleotide changes into the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNA,and/or by synthesis of the desired PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. Those skilled in theart will appreciate that amino acid changes may alter post-translationalprocesses of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, such as changing the number or positionof glycosylation sites or altering the membrane anchoringcharacteristics.

Variations in the native full-length sequence PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or invarious domains of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide described herein, can be made,for example, using any of the techniques and guidelines for conservativeand non-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide that results in a change in theamino acid sequence of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide as compared with the nativesequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Guidance in determining whichamino acid residue may be inserted, substituted or deleted withoutadversely affecting the desired activity may be found by comparing thesequence of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 fragments by enzymatic digestion,e.g., by treating the protein with an enzyme known to cleave proteins atsites defined by particular amino acid residues, or by digesting the DNAwith suitable restriction enzymes and isolating the desired fragment.Yet another suitable technique involves isolating and amplifying a DNAfragment encoding a desired polypeptide fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide fragments share at least one biologicaland/or immunological activity with the native PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide disclosedherein.

Conservative substitutions of interest are shown in Table 6 under theheading of preferred substitutions. If such substitutions result in achange in biological activity, then more substantial changes,denominated exemplary substitutions in Table 6, or as further describedbelow in reference to amino acid classes, are preferably introduced andthe products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg ®) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys ©) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in function or immunological identity of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

Amino acids may be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)(3) acidic: Asp (D), Glu (E)(4) basic: Lys (K), Arg (R), His (H)Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287 PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 Polypeptides

Covalent modifications of PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides are included within the scopeof this invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. Derivatization withbifunctional agents is useful, for instance, for crosslinking PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides to a water-insoluble support matrix or surface for use inthe method for purifying anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

Addition of glycosylation sites to the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide may be accomplishedby altering the amino acid sequence. The alteration may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 (for O-linkedglycosylation sites). The PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides comprises linkingthe PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides of the present invention may also bemodified in a way to form a chimeric molecule comprising the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide fused to another, heterologous polypeptide or amino acidsequence.

Such a chimeric molecule comprises a fusion of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidewith a tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino- or carboxyl-terminus of the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. The presence of suchepitope-tagged forms of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

The chimeric molecule may comprise a fusion of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidewith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide in place of at least one variable region within an Igmolecule. In a particularly preferred aspect of the invention, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 Polypeptides

The description below relates primarily to production of PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides byculturing cells transformed or transfected with a vector containingPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 nucleic acid. It is, of course, contemplated that alternativemethods, which are well known in the art, may be employed to preparePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides. For instance, the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 sequence, or portions thereof,may be produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide.

1. Isolation of DNA Encoding PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 Polypeptides

DNA encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides may be obtained from a cDNA libraryprepared from tissue believed to possess the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 mRNA and to express itat a detectable level. Accordingly, human PRO196-, PRO217-, PRO231-,PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-,PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-,PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709-or PRO779-DNA can be conveniently obtained from a cDNA library preparedfrom human tissue, such as described in the Examples. The PRO196-,PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-,PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-encoding gene may also be obtained from agenomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide or oligonucleotides of at least about 20-80 bases) designedto identify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT had; E. coli W3110 strain 37D6, which has thecomplete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvGkan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesare derived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/−DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesmay be inserted into a replicable vector for cloning (amplification ofthe DNA) or for expression. Various vectors are publicly available. Thevector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO196-, PRO217-, PRO231-, PRO236-,PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-,PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709- orPRO779-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-,PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-encoding nucleic acid, such as DHFR orthymidine kinase. An appropriate host cell when wild-type DHFR isemployed is the CHO cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). A suitable selection gene for use in yeast is the trp1gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature,282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al.,Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-,PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-,PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-,PRO7425-, PRO10102-, PRO10282-, PRO61709- or PRO779-encoding nucleicacid sequence to direct mRNA synthesis. Promoters recognized by avariety of potential host cells are well known. Promoters suitable foruse with prokaryotic hosts include the β-lactamase and lactose promotersystems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promotersystem [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], andhybrid promoters such as the tac promoter [deBoer et al., Proc. Natl.Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide by higher eukaryotesmay be increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides in recombinant vertebrate cell cultureare described in Gething et al., Nature, 293:620-625 (1981); Mantei etal., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides may be recovered from culture medium orfrom host cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidescan be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides from recombinant cell proteinsor polypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide. Various methods of protein purification may beemployed and such methods are known in the art and described for examplein Deutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptideproduced.

E. Uses for PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 Polypeptides

Nucleotide sequences (or their complement) encoding PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptideshave various applications in the art of molecular biology, includinguses as hybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 nucleic acid will also be usefulfor the preparation of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides by the recombinant techniquesdescribed herein.

The full-length native sequence PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene, or portions thereof, may be used ashybridization probes for a cDNA library to isolate the full-lengthPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 cDNA or to isolate still other cDNAs (for instance, thoseencoding naturally-occurring variants of PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides or PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptidesfrom other species) which have a desired sequence identity to the nativePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 sequence disclosed herein. Optionally, the length of the probeswill be about 20 to about 50 bases. The hybridization probes may bederived from at least partially novel regions of the full length nativenucleotide sequence wherein those regions may be determined withoutundue experimentation or from genomic sequences including promoters,enhancer elements and introns of native sequence PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. By way of example, ascreening method will comprise isolating the coding region of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 gene using the known DNA sequence to synthesize a selected probeof about 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene of the present invention can be usedto screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 nucleic acids include antisense or senseoligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 mRNA (sense) or PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNA(antisense) sequences. Antisense or sense oligonucleotides, according tothe present invention, comprise a fragment of the coding region ofPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. Antisense orsense oligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages, such as thosedescribed in WO 91/06629) and wherein such sugar linkages are resistantto endogenous nucleases. Such oligonucleotides with resistant sugarlinkages are stable in vivo (i.e., capable of resisting enzymaticdegradation) but retain sequence specificity to be able to bind totarget nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 coding sequences.

Nucleotide sequences encoding a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide can also be used to constructhybridization probes for mapping the gene which encodes that PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide and for the genetic analysis of individuals with geneticdisorders. The nucleotide sequences provided herein may be mapped to achromosome and specific regions of a chromosome using known techniques,such as in situ hybridization, linkage analysis against knownchromosomal markers, and hybridization screening with libraries.

When the coding sequences for PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 encode a protein which binds to anotherprotein (for example, where the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 is a receptor), the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide can be usedin assays to identify the other proteins or molecules involved in thebinding interaction. By such methods, inhibitors of the receptor/ligandbinding interaction can be identified. Proteins involved in such bindinginteractions can also be used to screen for peptide or small moleculeinhibitors or agonists of the binding interaction. Also, the receptorPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 can be used to isolate correlative ligand(s). Screening assayscan be designed to find lead compounds that mimic the biologicalactivity of a native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide or a receptor for PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides. Such screening assays will include assays amenable tohigh-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. The invention provides cDNA encoding a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide which can beused to clone genomic DNA encoding a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells which express DNA encoding PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides. Any technique known in the art may be used to introduce atarget gene transgene into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited topronuclear microinjection (U.S. Pat. Nos. 4,873,191, 4,736,866 and4,870,009); retrovirus mediated gene transfer into germ lines (Van derPutten, et al., Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); genetargeting in embryonic stem cells (Thompson, et al., Cell, 56:313-321(1989)); nonspecific insertional inactivation using a gene trap vector(U.S. Pat. No. 6,436,707); electroporation of embryos (Lo, Mol. Cell.Biol., 3:1803-1814 (1983)); and sperm-mediated gene transfer (Lavitrano,et al., Cell, 57:717-723 (1989)); etc. Typically, particular cells wouldbe targeted for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 transgene incorporation withtissue-specific enhancers. Transgenic animals that include a copy of atransgene encoding a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its overexpression.In accordance with this facet of the invention, an animal is treatedwith the reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.Alternatively, non-human homologues of PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides can be used toconstruct a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 “knock out” animal which has a defective or alteredgene encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 proteins as a result of homologous recombinationbetween the endogenous gene encoding PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides and altered genomicDNA encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides introduced into an embryonic stem cellof the animal. Preferably the knock out animal is a mammal. Morepreferably, the mammal is a rodent such as a rat or mouse. For example,cDNA encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides can be used to clone genomic DNAencoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides in accordance with establishedtechniques. A portion of the genomic DNA encoding the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide canbe deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the gene encoding the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.

In addition, knockout mice can be highly informative in the discovery ofgene function and pharmaceutical utility for a drug target, as well asin the determination of the potential on-target side effects associatedwith a given target. Gene function and physiology are so well conservedbetween mice and humans, since they are both mammals and contain similarnumbers of genes, which are highly conserved between the species. It hasrecently been well documented, for example, that 98% of genes on mousechromosome 16 have a human ortholog (Mural et al., Science 296:1661-71(2002)).

Although gene targeting in embryonic stem (ES) cells has enabled theconstruction of mice with null mutations in many genes associated withhuman disease, not all genetic diseases are attributable to nullmutations. One can design valuable mouse models of human diseases byestablishing a method for gene replacement (knock-in) which will disruptthe mouse locus and introduce a human counterpart with mutation,Subsequently one can conduct in vivo drug studies targeting the humanprotein (Kitamoto et. Al., Biochemical and Biophysical Res. Commun.,222:742-47 (1996)).

Nucleic acid encoding the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes and theisolated nucleic acid sequences may be used for recombinantly expressingthose markers.

The nucleic acid molecules encoding the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptides or fragments thereofdescribed herein are useful for chromosome identification. In thisregard, there exists an ongoing need to identify new chromosome markers,since relatively few chromosome marking reagents, based upon actualsequence data are presently available. Each PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 nucleic acid molecule ofthe present invention can be used as a chromosome marker.

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides and nucleic acid molecules of thepresent invention may also be used diagnostically for tissue typing,wherein the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides of the present invention may bedifferentially expressed in one tissue as compared to another,preferably in a diseased tissue as compared to a normal tissue of thesame tissue type. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides described herein may also be employed astherapeutic agents. The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide or agonist or antagonistthereof is employed, normal dosage amounts may vary from about 10 ng/kgto up to 100 mg/kg of mammal body weight or more per day, preferablyabout 1 μg/kg/day to 10 mg/kg/day, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. No.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, microencapsulation of the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide (agonists) or prevent theeffect of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide (antagonists). Agonists that mimic aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide would be especially valuable therapeutically in thoseinstances where a negative phenotype is observed based on findings withthe non-human transgenic animal whose genome comprises a disruption ofthe gene which encodes for the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Antagonists that prevent theeffects of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide would be especially valuabletherapeutically in those instances where a positive phenotype isobserved based upon observations with the non-human transgenic knockoutanimal. Screening assays for antagonist drug candidates are designed toidentify compounds that bind or complex with the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide encoded bythe genes identified herein, or otherwise interfere with the interactionof the encoded polypeptide with other cellular proteins. Such screeningassays will include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. The PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptideencoded by the gene identified herein or the drug candidate isimmobilized on a solid phase, e.g., on a microtiter plate, by covalentor non-covalent attachments. Non-covalent attachment generally isaccomplished by coating the solid surface with a solution of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide and drying. Alternatively, an immobilized antibody, e.g., amonoclonal antibody, specific for the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide to be immobilized canbe used to anchor it to a solid surface. The assay is performed byadding the non-immobilized component, which may be labeled by adetectable label, to the immobilized component, e.g., the coated surfacecontaining the anchored component. When the reaction is complete, thenon-reacted components are removed, e.g., by washing, and complexesanchored on the solid surface are detected. When the originallynon-immobilized component carries a detectable label, the detection oflabel immobilized on the surface indicates that complexing occurred.Where the originally non-immobilized component does not carry a label,complexing can be detected, for example, by using a labeled antibodyspecifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide may be added to a cell alongwith the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide indicates that the compound is anantagonist to the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Alternatively, antagonists maybe detected by combining the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide and a potential antagonist withmembrane-bound PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide receptors or recombinant receptors underappropriate conditions for a competitive inhibition assay. The PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide can be labeled, such as by radioactivity, such that thenumber of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide molecules bound to the receptor can beused to determine the effectiveness of the potential antagonist. Thegene encoding the receptor can be identified by numerous methods knownto those of skill in the art, for example, ligand panning and FACSsorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5(1991). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Transfected cells that aregrown on glass slides are exposed to labeled PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. The PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an interactive sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide can be photoaffinity-linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

Another approach in assessing the effect of an antagonist to a PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide, would be administering a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 antagonist to a wild-type mousein order to mimic a known knockout phenotype. Thus, one would initiallyknockout the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 gene of interest and observe the resultant phenotypeas a consequence of knocking out or disrupting the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene.Subsequently, one could then assess the effectiveness of an antagonistto the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide by administering an antagonist to thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide to a wild-type mouse. An effective antagonist wouldbe expected to mimic the phenotypic effect that was initially observedin the knockout animal.

Likewise, one could assess the effect of an agonist to a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, byadministering a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 agonist to a non-human transgenic mouse in order toameliorate a known negative knockout phenotype. Thus, one wouldinitially knockout the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 gene of interest and observe the resultantphenotype as a consequence of knocking out or disrupting the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene.Subsequently, one could then assess the effectiveness of an agonist tothe PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide by administering an agonist to thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide to a the non-human transgenic mouse. An effectiveagonist would be expected to ameliorate the negative phenotypic effectthat was initially observed in the knockout animal.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with a labeledPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide in the presence of the candidate compound. Theability of the compound to enhance or block this interaction could thenbe measured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.

Another potential PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide antagonist is an antisense RNAor DNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides herein, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide (antisense—Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide. When antisense DNA is used, oligodeoxyribonucleotidesderived from the translation-initiation site, e.g., between about −10and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, thereby blocking the normalbiological activity of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Diagnostic and therapeutic uses of the herein disclosed molecules mayalso be based upon the positive functional assay hits disclosed anddescribed below.

F. Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236, Anti-PRO245,Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328, Anti-PRO344,Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731, Anti-PRO732,Anti-PRO1003, Anti-PRO1104, Anti-PRO1151, Anti-PRO1244, Anti-PRO1298,Anti-PRO1313, Anti-PRO1570, Anti-PRO1886, Anti-PRO1891, Anti-PRO4409,Anti-PRO5725, Anti-PRO5994, Anti-PRO6097, Anti-PRO7425, Anti-PRO10102,Anti-PRO10282, Anti-PRO61709 or Anti-PRO779 Antibodies

The present invention provides anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibodies which may find use herein as therapeutic and/or diagnosticagents. Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g., by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

Monoclonal antibodies or antibody fragments can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies of the inventionmay further comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of a humanized anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody are contemplated. For example, the humanized antibody may be anantibody fragment, such as a Fab, which is optionally conjugated withone or more cytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized antibody may be an intact antibody, such asan intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 protein as described herein.Other such antibodies may combine a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 binding site with a binding sitefor another protein. Alternatively, an anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 arm may be combined with an arm which binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16), so as to focus and localize cellular defensemechanisms to the PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-,PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-,PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-,PRO7425-, PRO10102-, PRO10282-, PRO61709- or PRO779-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide. These antibodies possess aPRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-,PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,PRO10282-, PRO61709- or PRO779-binding arm and an arm which binds thecytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricinA chain, methotrexate or radioactive isotope hapten). Bispecificantibodies can be prepared as full length antibodies or antibodyfragments (e.g., F(ab′)₂ bispecific antibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463.U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificity (antibody-antigen combining sites) are fusedto immunoglobulin constant domain sequences. Preferably, the fusion iswith an Ig heavy chain constant domain, comprising at least part of thehinge, C_(H)2, and C_(H)3 regions. It is preferred to have the firstheavy-chain constant region (C_(H)1) containing the site necessary forlight chain bonding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. This providesfor greater flexibility in adjusting the mutual proportions of the threepolypeptide fragments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yield of the desiredbispecific antibody. It is, however, possible to insert the codingsequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chain combination.

The invention provides bispecific antibodies which are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a V_(H) connected to a V_(L) by a linker which is tooshort to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

8. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

9. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, agrowth inhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

The invention provides an anti-PRO196, anti-PRO217, anti-PRO231,anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779antibody (full length or fragments) which is conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236, Anti-PRO245,Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328, Anti-PRO344,Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731, Anti-PRO732,Anti-PRO1003, Anti-PRO1104, Anti-PRO1151, Anti-PRO1244, Anti-PRO1298,Anti-PRO1313, Anti-PRO1570, Anti-PRO1886, Anti-PRO1891, Anti-PRO4409,Anti-PRO5725, Anti-PRO5994, Anti-PRO6097, Anti-PRO7425, Anti-PRO10102,Anti-PRO10282, Anti-PRO61709 or Anti-PRO779 Antibody-MaytansinoidConjugates (Immunoconjugates)

Anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody-maytansinoidconjugates are prepared by chemically linking an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disufide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. The linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibody conjugated to one or morecalicheamicin molecules. The calicheamicin family of antibiotics arecapable of producing double-stranded DNA breaks at sub-picomolarconcentrations. For the preparation of conjugates of the calicheamicinfamily, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American CyanamidCompany). Structural analogues of calicheamicin which may be usedinclude, but are not limited to, γ₁ ¹, α₂ ¹, α₃ ¹, N-acetyl-γ₁ ¹, PSAGand θ¹ ₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode etal., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S.patents to American Cyanamid). Another anti-tumor drug that the antibodycan be conjugated is QFA which is an antifolate. Both calicheamicin andQFA have intracellular sites of action and do not readily cross theplasma membrane. Therefore, cellular uptake of these agents throughantibody mediated internalization greatly enhances their cytotoxiceffects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 antibodies of the invention include BCNU,streptozoicin, vincristine and 5-fluorouracil, the family of agentsknown collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the conjugate is used for diagnosis, it may comprise a radioactiveatom for scintigraphic studies, for example tc^(99m) or I¹²³, or a spinlabel for nuclear magnetic resonance (NMR) imaging (also known asmagnetic resonance imaging, mri), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-PRO196, anti-PRO217,anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709or anti-PRO779 antibody and cytotoxic agent may be made, e.g., byrecombinant techniques or peptide synthesis. The length of DNA maycomprise respective regions encoding the two portions of the conjugateeither adjacent one another or separated by a region encoding a linkerpeptide which does not destroy the desired properties of the conjugate.

The invention provides that the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

10. Immunoliposomes

The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies disclosed hereinmay also be formulated as immunoliposomes. A “liposome” is a smallvesicle composed of various types of lipids, phospholipids and/orsurfactant which is useful for delivery of a drug to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes. Liposomescontaining the antibody are prepared by methods known in the art, suchas described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S.Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23,1997. Liposomes with enhanced circulation time are disclosed in U.S.Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

11. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236,Anti-PRO245, Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328,Anti-PRO344, Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731,Anti-PRO732, Anti-PRO1003, Anti-PRO1104, Anti-PRO1151, Anti-PRO1244,Anti-PRO1298, Anti-PRO1313, Anti-PRO1570, Anti-PRO1886, Anti-PRO1891,Anti-PRO4409, Anti-PRO5725, Anti-PRO5994, Anti-PRO6097, Anti-PRO7425,Anti-PRO10102, Anti-PRO10282, Anti-PRO61709 or Anti-PRO779 Antibodies

The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies of the inventionhave various therapeutic and/or diagnostic utilities for a neurologicaldisorder; a cardiovascular, endothelial or angiogenic disorder; animmunological disorder; an oncological disorder; an embryonicdevelopmental disorder or lethality, or a metabolic abnormality. Forexample, anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodiesmay be used in diagnostic assays for PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779, e.g., detecting its expression(and in some cases, differential expression) in specific cells, tissues,or serum. Various diagnostic assay techniques known in the art may beused, such as competitive binding assays, direct or indirect sandwichassays and immunoprecipitation assays conducted in either heterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used inthe diagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies also are usefulfor the affinity purification of PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides from recombinant cell cultureor natural sources. In this process, the antibodies against PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides are immobilized on a suitable support, such a Sephadexresin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide to be purified, and thereafter the support is washedwith a suitable solvent that will remove substantially all the materialin the sample except the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)) asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a BLAST score of 70 (or insome cases 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones by Amylase Screening

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176-179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

(SEQ ID NO: 67) 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′The sequence of reverse oligonucleotide 2 was:

(SEQ ID NO: 68) 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′PCR was then performed as follows:

a. Denature 92° C., 5 minutes b.  3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c.  3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

Various polypeptide-encoding nucleic acid sequences were identified byapplying a proprietary signal sequence finding algorithm developed byGenentech, Inc. (South San Francisco, Calif.) upon ESTs as well asclustered and assembled EST fragments from public (e.g., GenBank) and/orprivate (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)databases. The signal sequence algorithm computes a secretion signalscore based on the character of the DNA nucleotides surrounding thefirst and optionally the second methionine codon(s) (ATG) at the 5′-endof the sequence or sequence fragment under consideration. Thenucleotides following the first ATG must code for at least 35unambiguous amino acids without any stop codons. If the first ATG hasthe required amino acids, the second is not examined. If neither meetsthe requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO6097,PRO7425, PRO10102, PRO10282, or PRO779 polypeptides as disclosed herein.These cDNAs were then deposited under the terms of the Budapest Treatywith the American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209, USA (ATCC) as shown in Table 7 below. Inaddition, the sequence of DNA98591 encoding PRO5994 polypeptides wasidentified from GenBank accession no.: AF048700; the sequence ofDNA347767 encoding PRO61709 polypeptides was identified from GenBankaccession no.: AB029000.

TABLE 7 Material ATCC Dep. No. Deposit Date DNA22779-1130 209280 Sep.18, 1997 DNA33094-1131 209256 Sep. 16, 1997 DNA34434-1139 209252 Sep.16, 1997 DNA35599-1168 209373 Oct. 16, 1997 DNA35638-1141 209265 Sep.16, 1997 DNA35639-1172 209396 Oct. 17, 1997 DNA35918-1174 209402 Oct.17, 1997 DNA39969-1185 209400 Oct. 17, 1997 DNA40587-1231 209438 Nov. 7,1997 DNA40592-1242 209492 Nov. 21, 1997 DNA44804-1248 209527 Dec. 10,1997 DNA44184-1319 209704 Mar. 26, 1998 DNA49631-1328 209806 Apr. 28,1998 DNA48331-1329 209715 Mar. 31, 1998 DNA48334-1435 209924 Jun. 2,1998 DNA58846-1409 209957 Jun. 9, 1998 DNA59616-1465 209991 Jun. 16,1998 DNA44694-1500 203114 Aug. 11, 1998 DNA64883-1526 203253 Sep. 9,1998 DNA66511-1563 203228 Sep. 15, 1998 DNA64966-1575 203575 Jan. 12,1999 DNA68885-1678 203311 Oct. 6, 1998 DNA80796-2523 203555 Dec. 22,1998 DNA76788-2526 203551 Dec. 22, 1998 DNA88004-2575 203890 Mar. 30,1999 DNA92265-2669 PTA-256 Jun. 22, 1999 DNA107701-2711 PTA-487 Aug. 3,1999 DNA108792-2753 PTA-617 Aug. 31, 1999 DNA129542-2808 PTA-1178 Jan.11, 2000 DNA148380-2827 PTA-1181 Jan. 11, 2000 DNA58801-1052  55820 Sep.5, 1996

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Example 4 Isolation of cDNA Clones Encoding Human PRO196 (NL1)Polypeptides [UNQ170]

NL1 was identified by screening the GenBank database using the computerprogram BLAST (Altshul et al., Methods in Enzymology 266:460-480(1996)). The NL1 sequence shows homology with known expressed sequencetag (EST) sequences T35448, T11442, and W77823. None of the known ESTsequences have been identified as full length sequences, or described asligands associate 1d with the TIE receptors.

Following its identification, NL1 was cloned from a human fetal lunglibrary prepared from mRNA purchased from Clontech, Inc. (Palo Alto,Calif., USA), catalog #6528-1, following the manufacturer'sinstructions.

The library was ligated into pRK5B vector, which is a precursor of pRK5Dthat does not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991). pRK5D, in turn, is a derivative of pRK5 (EP307,247, published 15 Mar. 1989), with minor differences within thepolylinker sequence. The library was screened by hybridization withsynthetic oligonucleotide probes:

NL1.5-1 SEQ. ID. NO: 69 5′-GCTGACGAACCAAGGCAACTACAAACTCCTGGT NL1.3-1SEQ. ID. NO: 70 5′-TGCGGCCGGACCAGTCCTCCATGGTCACCAGGAGTTTGTAG NL1.3-2SEQ. ID. NO: 71 5′-GGTGGTGAACTGCTTGCCGTTGTGCCATGTAAAbased on the ESTs found in the GenBank database. cDNA sequences weresequenced in their entireties.

The nucleotide and amino acid sequences of NL1 are shown in FIG. 1 (SEQ.ID. NO:1; DNA22779-1130) and FIG. 2 (SEQ. ID. NO: 2; PRO196),respectively.

NL1 shows a 23% sequence identity with both the TIE1 and the TIE2ligand.

A clone of NL1 (herein designated DNA22779-1130) was deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, on 18 Sep. 1997, under the terms of the BudapestTreaty, and has been assigned the deposit number ATCC 209280.

NL1 has been mapped to chromosome 9, band arm q13-q21.

Example 5 Isolation of cDNA Clones Encoding Human PRO217 Polypeptides[UNQ191]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isdesignated herein as DNA28760. Based on the assembled DNA28760 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO217.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer: 5′-AAAGACGCATCTGCGAGTGTCC-3′ (SEQ ID NO: 72)reverse PCR primer: 5′-TGCTGATTTCACACTGCTCTCCC-3′ (SEQ ID NO: 73)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA28760 consensus sequence which had the followingnucleotide sequence:

hybridization probe: (SEQ ID NO: 74)5′-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCTG CATC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO217 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue.

DNA sequencing of the isolated clones isolated as described above gavethe full-length DNA sequence for DNA33094-1131 [FIG. 3, SEQ ID NO:3];and the derived protein sequence for PRO217.

The entire coding sequence of DNA33094-1131 is included in FIG. 3 (SEQID NO:3). Clone DNA33094-1131 contains a single open reading frame withan apparent translational initiation site at nucleotide positions146-148, and an apparent stop codon at nucleotide positions 1283-1285.The predicted polypeptide precursor is 379 amino acids long with amolecular weight of approximately 41,528 daltons and an estimated pI ofabout 7.97. Analysis of the full-length PRO217 sequence shown in FIG. 4(SEQ ID NO:4) evidences the presence of a variety of importantpolypeptide domains, wherein the locations given for those importantpolypeptide domains are approximate as described above. Analysis of thefull-length PRO217 polypeptide shown in FIG. 4 evidences the presence ofthe following: a signal peptide from about amino acid 1 to about aminoacid 28; N-glycosylation sites from about amino acid 88 to about aminoacid 92, and from about amino acid 245 to about amino acid 249; atyrosine kinase phosphorylation site from about amino acid 370 to aboutamino acid 378; N-myristoylation sites from about amino acid 184 toabout amino acid 190, from about amino acid 185 to about amino acid 191,from about amino acid 189 to about amino acid 195, and from about aminoacid 315 to about amino acid 321; an ATP/GTP-binding site motif A(P-loop) from about amino acid 285 to about amino acid 293; and EGF-likedomain cysteine pattern signatures from about amino acid 198 to aboutamino acid 210, from about amino acid 230 to about amino acid 242, fromabout amino acid 262 to about amino acid 274, from about amino acid 294to about amino acid 306, and from about amino acid 326 to about aminoacid 338. Clone DNA33094-1131 has been deposited with the ATCC on Sep.16, 1997 and is assigned ATCC deposit no. 209256.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence shown in FIG. 4 (SEQ ID NO:4), PRO217 appears to bea novel EGF-like homologue.

Example 6 Isolation of cDNA Clones Encoding Human PRO231 Polypeptides[UNQ205]

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence was designated herein as DNA30933. Based on the DNA30933consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO231.

Three PCR primers (two forward and one reverse) were synthesized:

forward PCR primer 1 5′-CCAACTACCAAAGCTGCTGGAGCC-3′ (SEQ ID NO: 75)forward PCR primer 2 5′-GCAGCTCTATTACCACGGGAAGGA-3′ (SEQ ID NO: 76)reverse PCR primer 5′-TCCTTCCCGTGGTAATAGAGCTGC-3′ (SEQ ID NO: 77)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30933 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 78)5′-GGCAGAGAACCAGAGGCCGGAGGAGACTGCCTCTTTACAGCCAGG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO231 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO231 [herein designated as DNA34434-1139]and the derived protein sequence for PRO231.

The entire nucleotide sequence of DNA34434-1139 is shown in FIG. 5 (SEQID NO:5). Clone DNA34434-1139 contains a single open reading frame withan apparent translational initiation site at nucleotide positions173-175 and ending at the stop codon at nucleotide positions 1457-1459(FIG. 5; SEQ ID NO:5). The predicted polypeptide precursor is 428 aminoacids long (FIG. 6; SEQ ID NO:6). Clone DNA34434-1139 has been depositedwith ATCC on Sep. 16, 1997 and is assigned ATCC deposit no. ATCC 209252.

Analysis of the amino acid sequence of the full-length PRO231 suggeststhat it possesses 30% and 31% amino acid identity with the human and ratprostatic acid phosphatase precursor proteins, respectively.

Example 7 Isolation of cDNA Clones Encoding Human PRO236 Polypeptides[UNQ210]

Consensus DNA sequences were assembled relative to other EST sequencesusing pinup as described in Example 1 above. This consensus sequences isherein designated DNA30901. Based on the DNA30901 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO236. Basedupon the DNA30901 consensus sequence, a pair of PCR primers (forward andreverse) were synthesized:

forward PCR primer 5′-TGGCTACTCCAAGACCCTGGCATG-3′ (SEQ ID NO: 79)reverse PCR primer 5′-TGGACAAATCCCCTTGCTCAGCCC-3′ (SEQ ID NO: 80)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30901 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 81)5′-GGGCTTCACCGAAGCAGTGGACCTTTATTTTGACCACCTGATGTC CAGGG-3′

In order to screen several libraries for a source of full-length clones,DNA from the libraries was screened by PCR amplification with the PCRprimer pairs identified above. Positive libraries were then used toisolate clones encoding the PRO236 gene using the probe oligonucleotidesand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue for PRO236.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO236 [herein designated as DNA35599-1168](SEQ ID NO:7), the derived protein sequence for PRO236.

The entire nucleotide sequence of DNA35599-1168 is shown in FIG. 7 (SEQID NO:7). Clone DNA35599-1168 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 69-71and ending at the stop codon at nucleotide positions 1977-1979 (FIG. 7;SEQ ID NO:7). The predicted polypeptide precursor is 636 amino acidslong (FIG. 8; SEQ ID NO:8). Clone DNA35599-1168 has been deposited withATCC on Oct. 16, 1997 and is assigned ATCC deposit no. ATCC 209373.

Analysis of the amino acid sequence of the full-length PRO236polypeptide suggests that portions of this polypeptide possessessignificant homology to beta-galactosidase proteins derived from varioussources, thereby indicating that PRO236 may be a novelbeta-galactosidase homolog.

Example 8 Isolation of cDNA Clones Encoding Human PRO245 Polypeptides[UNO219]

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA30954.

Based on the DNA30954 consensus sequence, oligonucleotides weresynthesized to identify by PCR a cDNA library that contained thesequence of interest and for use as probes to isolate a clone of thefull-length coding sequence for PRO245.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-ATCGTTGTGAAGTTAGTGCCCC-3′ (SEQ ID NO: 82)reverse PCR primer 5′-ACCTGCGATATCCAACAGAATTG-3′ (SEQ ID NO: 83)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30954 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 84)5′-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAG TTCC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO245 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO245 [herein designated asDNA35638-1141] and the derived protein sequence for PRO245.

The entire nucleotide sequence of DNA35638-1141 is shown in FIG. 9 (SEQID NO:9). Clone DNA35638-1141 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 89-91and ending at the stop codon at nucleotide positions 1025-1027 (FIG. 9;SEQ ID NO:9). The predicted polypeptide precursor is 312 amino acidslong (FIG. 10; SEQ ID NO:10). Clone DNA35638-1141 has been depositedwith ATCC on Sep. 16, 1997 and is assigned ATCC deposit no. ATCC 209265.

Analysis of the amino acid sequence of the full-length PRO245 suggeststhat a portion of it possesses 60% amino acid identity with the humanc-myb protein and, therefore, may be a new member of the transmembraneprotein receptor tyrosine kinase family.

Example 9 Isolation of cDNA Clones Encoding Human PRO246 Polypeptides[UNQ220]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30955. Based on the DNA30955 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO246.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AGGGTCTCCAGGAGAAAGACTC-3′ (SEQ ID NO: 85)reverse PCR primer  5′-ATTGTGGGCCTTGCAGACATAGAC-3′ (SEQ ID NO: 86)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30955 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 87)5′-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCA GCTCC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO246 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO246 [herein designated asDNA35639-1172] (SEQ ID NO:11) and the derived protein sequence forPRO246.

The entire nucleotide sequence of DNA35639-1172 is shown in FIG. 11 (SEQID NO:11). Clone DNA35639-1172 contains a single open reading frame withan apparent translational initiation site at nucleotide positions126-128 and ending at the stop codon at nucleotide positions 1296-1298(FIG. 11). The predicted polypeptide precursor is 390 amino acids long(FIG. 12; SEQ ID NO:12). Clone DNA35639-1172 has been deposited withATCC on Oct. 17, 1997 and is assigned ATCC deposit no. ATCC 209396.

Analysis of the amino acid sequence of the full-length PRO246polypeptide suggests that it possess significant homology to the humancell surface protein HCAR, thereby indicating that PRO246 may be a novelcell surface virus receptor.

Example 10 Isolation of cDNA Clones Encoding Human PRO258 Polypeptides[UNQ225]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28746.

Based on the DNA28746 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO258.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GCTAGGAATTCCACAGAAGCCC-3′ (SEQ ID NO: 88)reverse PCR primer 5′-AACCTGGAATGTCACCGAGCTG-3′ (SEQ ID NO: 89)reverse PCR primer 5′-CCTAGCACAGTGACGAGGGACTTGGC-3′ (SEQ ID NO: 90)Additionally, synthetic oligonucleotide hybridization probes wereconstructed from the consensus DNA28740 sequence which had the followingnucleotide sequence:

hybridization probe (SEQ ID NO: 91)5′-AAGACACAGCCACCCTAAACTGTCAGTCTTCTGGGAGCAAGCCTG CAGCC-3′(SEQ ID NO: 92) 5′-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCAATCTTCACTATGCCTGT-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO258 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO258 [herein designated asDNA35918-1174] (SEQ ID NO:13) and the derived protein sequence forPRO258.

The entire nucleotide sequence of DNA35918-1174 is shown in FIG. 13 (SEQID NO:13). Clone DNA35918-1174 contains a single open reading frame withan apparent translational initiation site at nucleotide positions147-149 of SEQ ID NO:13 and ending at the stop codon after nucleotideposition 1340 of SEQ ID NO:13 (FIG. 13). The predicted polypeptideprecursor is 398 amino acids long (FIG. 14; SEQ ID NO:14). CloneDNA35918-1174 has been deposited with ATCC on Oct. 17, 1997 and isassigned ATCC deposit no. ATCC 209402.

Analysis of the amino acid sequence of the full-length PRO258polypeptide suggests that portions of it possess significant homology tothe CRTAM and the poliovirus receptor and have an Ig domain, therebyindicating that PRO258 is a new member of the Ig superfamily.

Example 11 Isolation of cDNA Clones Encoding Human PRO287 Polypeptides[UNQ250]

A consensus DNA sequence encoding PRO287 was assembled relative to theother identified EST sequences as described in Example 1 above, whereinthe consensus sequence is designated herein as DNA28728. Based on theDNA28728 consensus sequence, oligonucleotides were synthesized toidentify by PCR a cDNA library that contained the sequence of interestand for use as probes to isolate a clone of the full-length codingsequence for PRO287.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCGATTCATAGACCTCGAGAGT-3′ (SEQ ID NO: 93)reverse PCR primer 5′-GTCAAGGAGTCCTCCACAATAC-3′ (SEQ ID NO: 94)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28728 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 95)5′-GTGTACAATGGCCATGCCAATGGCCAGCGCATTGGCCGCTTC TGT-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO287 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO287 [herein designated as DNA39969-1185,SEQ ID NO:15] and the derived protein sequence for PRO287.

The entire nucleotide sequence of DNA39969-1185 is shown in FIG. 15 (SEQID NO:15). Clone DNA39969-1185 contains a single open reading frame withan apparent translational initiation site at nucleotide positions307-309 and ending at the stop codon at nucleotide positions 1552-1554(FIG. 15; SEQ ID NO:15). The predicted polypeptide precursor is 415amino acids long (FIG. 16; SEQ ID NO:16). Clone DNA39969-1185 has beendeposited with ATCC on Oct. 17, 1997 and is assigned ATCC deposit no.ATCC 209400.

Analysis of the amino acid sequence of the full-length PRO287 suggeststhat it may possess one or more procollagen C-proteinase enhancerprotein precursor or procollagen C-proteinase enhancer protein-likedomains. Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO287 shows nucleic acid sequence identity toprocollagen C-proteinase enhancer protein precursor and procollagenC-proteinase enhancer protein (47 and 54%, respectively).

Example 12 Isolation of cDNA Clones Encoding Human PRO328 Polypeptides[UNQ289]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35615. Based on the DNA35615 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO328.

Forward and reverse PCR primers were synthesized:

forward PCR primer 5′-TCCTGCAGTTTCCTGATGC-3′ (SEQ ID NO: 96)reverse PCR primer 5′-CTCATATTGCACACCAGTAATTCG-3′ (SEQ ID NO: 97)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35615 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 98)5′-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO328 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO328 [herein designated as DNA40587-1231](SEQ ID NO:17) and the derived protein sequence for PRO328.

The entire nucleotide sequence of DNA40587-1231 is shown in FIG. 17 (SEQID NO:17). Clone DNA40587-1231 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 15-17and ending at the stop codon at nucleotide positions 1404-1406 (FIG.17). The predicted polypeptide precursor is 463 amino acids long (FIG.18; SEQ ID NO:18). Clone DNA40587-1231 has been deposited with ATCC onNov. 7, 1997 and is assigned ATCC deposit no. ATCC 209438.

Analysis of the amino acid sequence of the full-length PRO328polypeptide suggests that portions of it possess significant homology tothe human glioblastoma protein and to the cysteine rich secretoryprotein thereby indicating that PRO328 may be a novel glioblastomaprotein or cysteine rich secretory protein.

Example 13 Isolation of cDNA Clones Encoding Human PRO344 Polypeptides[UNQ303]

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA34398. Based on the DNA34398 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO344.

Based on the DNA34398 consensus sequence, forward and reverse PCRprimers were synthesized as follows:

forward PCR primer(3439811) 5′-TACAGGCCCAGTCAGGACCAGGGG-3′(SEQ ID NO: 99) forward PCR primer(34398.f2) 5′-AGCCAGCCTCGCTCTCGG-3′(SEQ ID NO: 100) forward PCR primer(34398.f3) 5′-GTCTGCGATCAGGTCTGG-3′(SEQ ID NO: 101) reverse PCR primer(34398.r1) 5′-GAAAGAGGCAATGGATTCGC-3′(SEQ ID NO: 102) reverse PCR primer(34398.r2)5′-GACTTACACTTGCCAGCACAGCAC-3′ (SEQ ID NO: 103)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA34398 consensus sequence which had the followingnucleotide sequence

hybridization probe (34398.p1) (SEQ ID NO: 104)5′-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO344 genes using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO344 [herein designated as DNA40592-1242](SEQ ID NO:19) and the derived protein sequence for PRO344.

The entire nucleotide sequence of DNA40592-1242 is shown in FIG. 19 (SEQID NO:19). Clone DNA40592-1242 contains a single open reading frame withan apparent translational initiation site at nucleotide positions227-229 and ending at the stop codon at nucleotide positions 956-958(FIG. 19). The predicted polypeptide precursor is 243 amino acids long(FIG. 20; SEQ ID NO:20). Important regions of the native PRO344 aminoacid sequence include the signal peptide, the start of the matureprotein, and two potential N-myristoylation sites as shown in FIG. 20.Clone DNA40592-1242 has been deposited with the ATCC on Nov. 21, 1997and is assigned ATCC deposit no. ATCC 209492.

Analysis of the amino acid sequence of the full-length PRO344polypeptides suggests that portions of them possess significant homologyto various human and murine complement proteins, thereby indicating thatPRO344 may be a novel complement protein.

Example 14 Isolation of cDNA Clones Encoding Human PRO357 Polypeptides[UNQ314]

The sequence expression tag clone no. “2452972” by IncytePharmaceuticals, Palo Alto, Calif. was used to begin a data base search.The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases which overlapped with a portion of IncyteEST clone no. “2452972”. The EST databases included public EST databases(e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6frame translation of the EST sequence. Those comparisons resulting in aBLAST score of 70 (or in some cases 90) or greater that did not encodeknown proteins were clustered and assembled into consensus DNA sequenceswith the program “phrap” (Phil Green, University of Washington, Seattle,Wash.).

A consensus DNA sequence was then assembled relative to other ESTsequences using phrap. This consensus sequence is herein designatedDNA37162. In this case, the consensus DNA sequence was extended usingrepeated cycles of BLAST and phrap to extend the consensus sequence asfar as possible using the sources of EST sequences discussed above.

Based on the DNA37162 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO357. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as ber Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

PCR primers were synthesized as follows:

forward primer 1: 5′-CCCTCCACTGCCCCACCGACTG-3′; (SEQ ID NO: 105)reverse primer 1: 5′-CGGTTCTGGGGACGTTAGGGCTCG-3′; (SEQ ID NO: 106) andforward primer 2: 5′-CTGCCCACCGTCCACCTGCCTCAAT-3′. (SEQ ID NO: 107)Additionally, two synthetic oligonucleotide hybridization probes wereconstructed from the consensus DNA37162 sequence which had the followingnucleotide sequences:

hybridization probe 1: (SEQ ID NO: 108)5′-AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCA CC-3′; andhybridization probe 2: (SEQ ID NO: 109)5′-ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTG GG-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with aPCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO357 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith olio dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO357 [herein designated as DNA44804-1248](SEQ ID NO:21) and the derived protein sequence for PRO357.

The entire nucleotide sequence of DNA44804-1248 is shown in FIG. 21 (SEQID NO:21). Clone DNA44804-1248 contains a single open reading frame withan apparent translational initiation site at nucleotide positions137-139 and ending at the stop codon at nucleotide positions 1931-1933(FIG. 21). The predicted polypeptide precursor is 598 amino acids long(FIG. 22; SEQ ID NO:22). Clone DNA44804-1248 has been deposited withATCC on Dec. 10, 1997 and is assigned ATCC deposit no. ATCC 209527.

Analysis of the amino acid sequence of the full-length PRO357polypeptide therefore suggests that portions of it possess significanthomology to ALS, thereby indicating that PRO357 may be a novel leucinerich repeat protein related to ALS.

Example 15 Isolation of cDNA Clones Encoding Human PRO526 Polypeptides[UNQ330]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39626. Based on the DNA39626 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO526.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGGCTGCCCTGCAGTACCTCTACC-3′; (SEQ ID NO: 110)reverse PCR primer 5′-CCCTGCAGGTCATTGGCAGCTAGG-3′. (SEQ ID NO: 111)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA39626 consensus sequence which had the followingnucleotide sequence:

hybridization probe (SEQ ID NO: 112)5′-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCAC AC-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO526 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO526 [herein designated as UNQ330(DNA44184-1319)] (SEQ ID NO:23) and the derived protein sequence forPRO526.

The entire nucleotide sequence of UNQ330 (DNA44184-1319) is shown inFIG. 23 (SEQ ID NO:23). Clone UNQ330 (DNA44184-1319) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 514-516 and ending at the stop codon at nucleotidepositions 1933-1935 (FIG. 23). The predicted polypeptide precursor is473 amino acids long (FIG. 24; SEQ ID NO:24). The full-length PRO526protein shown in FIG. 24 has an estimated molecular weight of about50,708 daltons and a pI of about 9.28. Clone UNQ330 (DNA44184-1319) hasbeen deposited with the ATCC on Mar. 26, 1998 under ATCC accession no:209704. It is understood that the clone contains the actual sequence,whereas the sequences presented herein are representative based oncurrent sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO526polypeptide suggests that portions of it possess significant homology tothe leucine repeat rich proteins including ALS, SLIT, carboxypeptidaseand platelet glycoprotein V thereby indicating that PRO526 is a novelprotein which is involved in protein-protein interactions.

Still analyzing SEQ ID NO:24, the signal peptide sequence is at aboutamino acids 1-26. A leucine zipper pattern is at about amino acids135-156. A glycosaminoglycan attachment is at about amino acids 436-439.N-glycosylation sites are at about amino acids 82-85, 179-182, 237-240and 423-426. A von Willebrand factor (VWF) type C domain(s) is found atabout amino acids 411-425. The skilled artisan can understand whichnucleotides correspond to these amino acids based on the sequencesprovided herein.

Example 16 Isolation of cDNA Clones Encoding Human PRO724 Polypeptides[UNQ389]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35603. Based on the DNA35603 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO724.

Pairs of PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 5′-GGCTGTCACTGTGGAGACAC-3′ (SEQ ID NO: 113)forward PCR primer 2 5′-GCAAGGTCATTACAGCTG-3′ (SEQ ID NO: 114)reverse PCR primer 1 5′-AGAACATAGGAGCAGTCCCACTC-3′ (SEQ ID NO: 115)reverse PCR primer 2 5′-TGCCTGCTGCTGCACAATCTCAG-3′ (SEQ ID NO: 116)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35603 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 117)5′-GGCTATTGCTTGCCTTGGGACAGACCCTGTGGCTTAGGCTCTGGC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO724 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO724 [herein designated as UNQ389(DNA49631-1328)] (SEQ ID NO:25) and the derived protein sequence forPRO724.

The entire nucleotide sequence of UNQ389 (DNA49631-1328) is shown inFIG. 25 (SEQ ID NO:25). Clone UNQ389 (DNA49631-1328) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 546-548 and ending at the stop codon at nucleotidepositions 2685-2687 (FIG. 25). The predicted polypeptide precursor is713 amino acids long (FIG. 26; SEQ ID NO:26). The full-length PRO724protein shown in FIG. 26 has an estimated molecular weight of about76,193 daltons and a pI of about 5.42. Analysis of the full-lengthPRO724 amino acid sequence shown in FIG. 26 (SEQ ID NO:26) evidences thepresence of the following: a signal peptide from about amino acid 1 toabout amino acid 16, a transmembrane domain from about amino acid 442 toabout amino acid 462 and LDL receptor class A domain regions from aboutamino acid 152 to about amino acid 171, about amino acid 331 to aboutamino acid 350, about amino acid 374 to about amino acid 393 and aboutamino acid 411 to about amino acid 430. Clone UNQ389 (DNA49631-1328) hasbeen deposited with ATCC on Apr. 28, 1998 and is assigned ATCC depositno. 209806.

Analysis of the amino acid sequence of the full-length PRO724polypeptide suggests that it possesses significant sequence similarityto the human LDL receptor protein, thereby indicating that PRO724 may bea novel LDL receptor homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO724 amino acid sequence and the followingDayhoff sequences, P_R48547, MMAM2R_(—)1, LRP2_RAT, P_R60517, P_R47861,P_R05533, A44513_(—)1, A30363, P_R74692 and LMLIPOPHO_(—)1.

Example 17 Isolation of cDNA Clones Encoding Human PRO731 Polypeptides[UNQ395]

A database was used to search expressed sequence tag (EST) databases.The EST database used herein was the proprietary EST DNA databaseLIFESEQ®, of Incyte Pharmaceuticals, Palo Alto, Calif. Incyte clone2581326 was herein identified and termed DNA42801. Based on the DNA42801sequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO731.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GTAAGCACATGCCTCCAGAGGTGC-3′; (SEQ ID NO: 118)reverse PCR primer 5′-GTGACGTGGATGCTTGGGATGTTG-3′. (SEQ ID NO: 119)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA42801 sequence which had the followingnucleotide sequence:

hybridization probe (SEQ ID NO: 120)5′-TGGACACCTTCAGTATTGATGCCAAGACAGGCCAGGTCATTCTGC GTCGA-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO731 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255). The cDNA librariesused to isolate the cDNA clones were constructed by standard methodsusing commercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with olio dT containing a NotI site,linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the SfiI site; see, Holmeset al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotIsites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO731 [herein designated as UNQ395(DNA48331-1329)] (SEQ ID NO:27) and the derived protein sequence forPRO731.

The entire nucleotide sequence of UNQ395 (DNA48331-1329) is shown inFIG. 27 (SEQ ID NO:27). Clone UNQ395 (DNA48331-1329) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 329-331 and ending at the stop codon at nucleotidepositions 3881-3883 (FIG. 27). The predicted polypeptide precursor is1184 amino acids long (FIG. 28; SEQ ID NO:28). The full-length PRO731protein shown in FIG. 28 has an estimated molecular weight of about129,022 daltons and a pI of about 5.2. Clone UNQ395 (DNA48331-1329) wasdeposited with the ATCC on Mar. 31, 1998 under ATCC accession no:209715. Regarding the sequence, it is understood that the depositedclone contains the correct sequence, and the sequences provided hereinare based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO731polypeptide suggests that portions of it possess significant identityand similarity to members of the protocadherin family, therebyindicating that PRO731 may be a novel protocadherin.

Still analyzing the amino acid sequence of SEQ ID NO:28, the putativesignal peptide is at about amino acids 1-13 of SEQ ID NO:28. Thetransmembrane domain is at amino acids 719-739 of SEQ ID NO:28. TheN-glycosylation of SEQ ID NO:28 are as follows: 415-418, 582-586,659-662, 662-665, and 857-860. The cadherin extracellular repeateddomain signatures are at about amino acids (of SEQ ID NO:28): 123-133,232-242, 340-350, 448-458, and 553-563. The corresponding nucleotidescan be routinely determined given the sequences provided herein.

Example 18 Isolation of cDNA Clones Encoding Human PRO732 Polypeptides[UNQ0396]

A yeast screening assay was employed to identify cDNA clones thatencoded potential secreted proteins. Use of this yeast screening assayallowed identification of a single cDNA clone whose sequence (hereindesignated as DNA42580). The DNA42580 sequence was then compared to avariety of known EST sequences to identify homologies. The EST databasesemployed included public EST databases (e.g., GenBank) and a proprietaryEST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.).The search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

Using the above analysis, a consensus DNA sequence was assembledrelative to other EST sequences using phrap. This consensus sequence isherein designated consen01. Proprietary Genentech EST sequences wereemployed in the consensus assembly and they are herein designatedDNA20239, DNA38050 and DNA40683.

Based on the consen01 sequence, oligonucleotides were synthesized: 1) toidentify by PCR a cDNA library that contained the sequence of interest,and 2) for use as probes to isolate a clone of the full-length codingsequence for PRO732. Forward and reverse PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100-1000 bp in length. The probe sequences are typically 40-55bp in length. In some cases, additional oligonucleotides are synthesizedwhen the consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-ATGTTTGTGTGGAAGTGCCCCG-3′ (SEQ ID NO: 121)forward PCR primer 5′-GTCAACATGCTCCTCTGC-3′ (SEQ ID NO: 122)reverse PCR primer 5′-AATCCATTGTGCACTGCAGCTCTAGG-3′ (SEQ ID NO: 123)reverse PCR primer 5′-GAGCATGCCACCACTGGACTGAC-3′ (SEQ ID NO: 124)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA44143 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 125)5′-GCCGATGCTGTCCTAGTGGAAACAACTCCACTGTAACTAGATTGA TCTATGCAC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO732 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue (LIB26). The cDNA libraries used to isolate the cDNA cloneswere constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with olio dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 88-90 and ending at the stop codon found at nucleotidepositions 1447-1449 (FIG. 29, SEQ ID NO:29). The predicted polypeptideprecursor is 453 amino acids long, has a calculated molecular weight ofapproximately 50,419 daltons and an estimated pI of approximately 5.78.Analysis of the full-length PRO732 sequence shown in FIG. 30 (SEQ IDNO:30) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 28, transmembrane domains fromabout amino acid 37 to about amino acid 57, from about amino acid 93 toabout amino acid 109, from about amino acid 126 to about amino acid 148,from about amino acid 151 to about amino acid 172, from about amino acid197 to about amino acid 215, from about amino acid 231 to about aminoacid 245, from about amino acid 260 to about amino acid 279, from aboutamino acid 315 to about amino acid 333, from about amino acid 384 toabout amino acid 403 and from about amino acid 422 to about amino acid447, potential N-glycosylation sites from about amino acid 33 to aboutamino acid 36, from about amino acid 34 to about amino acid 37, fromabout amino acid 179 to about amino acid 183, from about amino acid 298to about amino acid 301, from about amino acid 337 to about amino acid340 and from about amino acid 406 to about amino acid 409, an amino acidblock having homology to the MIP family of proteins from about aminoacid 119 to about amino acid 149 and an amino acid block having homologyto DNA/RNA non-specific endonuclease proteins from about amino acid 279to about amino acid 286. Clone DNA48334-1435 has been deposited withATCC on Jun. 2, 1998 and is assigned ATCC deposit no. 209924.

Analysis of the amino acid sequence of the full-length PRO732polypeptide suggests that it possesses significant sequence similarityto the Diff33 protein, thereby indicating that PRO732 may be a novelDiff33 homolog. More specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35) evidenced significant homology between thePRO732 amino acid sequence and the following Dayhoff sequences,HS179M20_(—)2, MUSTETU_(—)1, CER11H6_(—)2, RATDRP_(—)1, S51256, E69226,AE000869_(—)1, JC4120, CYB_PARTE and P_R50619.

Example 19 Isolation of cDNA Clones Encoding Human PRO1003 Polypeptides[UNQ487]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single Incyte EST cluster sequencedesignated herein as 43055. This sequence was then compared to a varietyof EST databases which included public EST databases (e.g., GenBank) anda proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, PaloAlto, Calif.) to identify existing homologies. The homology search wasperformed using the computer program BLAST or BLAST2 (Altshul et al.,Methods in Enzymology 266:460-480 (1996)). Those comparisons resultingin a BLAST score of 70 (or in some cases 90) or greater that did notencode known proteins were clustered and assembled into a consensus DNAsequence with the program “phrap” (Phil Green, University of Washington,Seattle, Wash.). The consensus sequence obtained therefrom is hereindesignated consen01.

In light of an observed sequence homology between the consensus sequenceand an EST sequence encompassed within the Incyte EST clone no. 2849382,the Incyte EST clone 2849382 was purchased and the cDNA insert wasobtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.31.

The entire nucleotide sequence of DNA58846-1409 is shown in FIG. 31 (SEQID NO:31). Clone DNA58846-1409 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 41-43and ending at the stop codon at nucleotide positions 293-295 (FIG. 31).The predicted polypeptide precursor is 84 amino acids long (FIG. 32; SEQID NO:32). The full-length PRO1003 protein shown in FIG. 32 has anestimated molecular weight of about 9,408 daltons and a pI of about9.28. Analysis of the full-length PRO1003 sequence shown in FIG. 32 (SEQID NO:32) evidences the presence of a signal peptide at amino acids 1 toabout 24, and a cAMP- and cGMP-dependent protein kinase phosphorylationsite at about amino acids 58 to about 61. Analysis of the amino acidsequence of the full-length PRO1003 polypeptide using the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced homology between thePRO1003 amino acid sequence and the following Dayhoff sequences:AOPCZA363_(—)3, SRTX_ATREN, A48298, MHVJHMS_(—)1, VGL2_CVMJH,DHDHTC2_(—)2, CORT_RAT, TAL6_HUMAN, P_W14123, and DVUFI_(—)2.

Example 20 Isolation of cDNA Clones Encoding Human PRO1104 Polypeptides[UNQ547]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (Lifeseq®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56446.

In light of an observed sequence homology between the DNA56446 sequenceand an EST sequence encompassed within the Incyte EST clone no. 2837496,the Incyte EST clone 2837496 was purchased and the cDNA insert wasobtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.33 and is herein designated as DNA59616-1465.

The entire nucleotide sequence of DNA59616-1465 is shown in FIG. 33 (SEQID NO:33). Clone DNA59616-1465 contains a single open reading frame withan apparent translational initiation site at nucleotide positions109-111 and ending at the stop codon at nucleotide positions 1132-1134of SEQ ID NO:33 (FIG. 33). The predicted polypeptide precursor is 341amino acids long (FIG. 34; SEQ ID NO:34). The full-length PRO1104protein shown in FIG. 34 has an estimated molecular weight of about36,769 daltons and a pI of about 9.03. Clone DNA59616-1465 has beendeposited with ATCC on Jun. 16, 1998 under ATCC accession no: 209991. Itis understood that the deposited clone has the actual nucleic acidsequence and that the sequences provided herein are based on knownsequencing techniques.

Analyzing FIG. 34, a signal peptide is at about amino acids 1-22 of SEQID NO:34. N-myristoylation sites are at about amino acids 41-46,110-115, 133-138, 167-172 and 179-184 of SEQ ID NO:34.

Example 21 Isolation of cDNA Clones Encoding Human PRO1151 Polypeptides[UNQ581]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA40665. Based on the DNA40665 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1151.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCAGACGCTGCTCTTCGAAAGGGTC-3′ (SEQ ID NO: 126)reverse PCR primer 5′-GGTCCCCGTAGGCCAGGTCCAGC-3′ (SEQ ID NO: 127)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA40665 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 128)5′-CTACTTCTTCAGCCTCAATGTGCACAGCTGGAATTACAAGGAGAC GTACG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1151 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1151 (designated herein as DNA44694-1500[FIG. 35, SEQ ID NO:35]; and the derived protein sequence for PRO1151.

The entire nucleotide sequence of DNA44694-1500 is shown in FIG. 35 (SEQID NO:35). Clone DNA44694-1500 contains a single open reading frame withan apparent translational initiation site at nucleotide positions272-274 and ending at the stop codon at nucleotide positions 1049-1051(FIG. 35). The predicted polypeptide precursor is 259 amino acids long(FIG. 36; SEQ ID NO:36). The full-length PRO1151 protein shown in FIG.36 has an estimated molecular weight of about 28,770 daltons and a pI ofabout 6.12. Analysis of the full-length PRO1151 sequence shown in FIG.36 (SEQ ID NO:36) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 20, a potentialN-glycosylation site from about amino acid 72 to about amino acid 75 andamino acid sequence blocks having homology to C1q domain-containingproteins from about amino acid 144 to about amino acid 178, from aboutamino acid 78 to about amino acid 111 and from about amino acid 84 toabout amino acid 117. Clone UNQ581 (DNA44694-1500) has been depositedwith ATCC on Aug. 11, 1998 and is assigned ATCC deposit no. 203114.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST-2 sequence alignment analysis of the full-length sequenceshown in FIG. 36 (SEQ ID NO:36), evidenced significant homology betweenthe PRO1151 amino acid sequence and the following Dayhoff sequences:ACR3_HUMAN, HP25_TAMAS, HUMC1QB2_(—)1, P_R99306, CA1F_HUMAN, JX0369,CA24_HUMAN, S32436, P_R28916 and CA54_HUMAN.

Example 22 Isolation of cDNA Clones Encoding Human PRO1244 Polypeptides[UNQ628]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the LIFESEQ®database, designated cluster no. 7874. This EST cluster sequence wasthen compared to a variety of expressed sequence tag (EST) databaseswhich included public EST databases (e.g., GenBank) and a proprietaryEST DNA databases (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.;Genentech, South San Francisco, Calif.) to identify existing homologies.One or more of the ESTs was derived from a library constructed fromtissue of the corpus cavernosum. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases 90) or greater that did not encode knownproteins were clustered and assembled into a consensus DNA sequence withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designated“DNA56011”.

In light of the sequence homology between the DNA56011 sequence and anEST sequence contained within Incyte EST No. 3202349, the EST clone no.3202349 was purchased and the cDNA insert was obtained and sequenced.The sequence of this cDNA insert is shown in FIG. 37 (SEQ ID NO:37) andis herein designated “DNA64883-1526”.

The full length clone shown in FIG. 37 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 9-11 and ending at the stop codon found at nucleotidepositions 1014-1016 (FIG. 37; SEQ ID NO:37). The predicted polypeptideprecursor (FIG. 38, SEQ ID NO:38) is 335 amino acids long. PRO1244 has acalculated molecular weight of approximately 38,037 daltons and anestimated pI of approximately 9.87. Other features include a signalpeptide at about amino acids 1-29; transmembrane domains at about aminoacids 183-205, 217-237, 271-287, and 301-321; potential N-glycosylationsites at about amino acids 71-74, and 215-218; and a cell attachmentsequence at about amino acids 150-152.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 38 (SEQ ID NO:38), revealed homology between the PRO1244amino acid sequence and the following Dayhoff sequences: AF008554_(—)1,P_(—)485334, G02297, HUMN33S11_(—)1, HUMN33S10_(—)1, YO13_CAEEL,GEN13255, S49758, E70107, and ERP5_MEDSA.

Clone DNA64883-1526 was deposited with the ATCC on Sep. 9, 1998, and isassigned ATCC deposit no. 203253.

Example 23 Isolation of cDNA Clones Encoding Human PRO1298 Polypeptides[UNQ666]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from an Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. One or more of the ESTs was derived from a diseased prostatetissue library. The homology search was performed using the computerprogram BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70(or in some cases 90) or greater that did not encode known proteins wereclustered and assembled into a consensus DNA sequence with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Theconsensus sequence obtained therefrom is herein designated DNA56389.

In light of the sequence homology between the DNA56389 sequence and anEST sequence contained within an Incyte EST within the assembly fromwith the consensus sequence was derived, Incyte clone 3355717 waspurchased and the cDNA insert was obtained and sequenced. The sequenceof this cDNA insert is shown in FIG. 39 and is herein designated asDNA66511-1563.

The full length clone shown in FIG. 39 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 94-96 and ending at the stop codon found at nucleotidepositions 1063-1065 (FIG. 39; SEQ ID NO:39). The predicted polypeptideprecursor (FIG. 40, SEQ ID NO:40) is 323 amino acids long. The signalpeptide is at about amino acids 1-15 of SEQ ID NO:40. PRO1298 has acalculated molecular weight of approximately 37,017 daltons and anestimated pI of approximately 8.83. Clone DNA66511-1563 was depositedwith the ATCC on Sep. 15, 1998 and is assigned ATCC deposit no. 203228.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 40 (SEQ ID NO:40), revealed sequence identity between thePRO1298 amino acid sequence and the following Dayhoff sequences (dataincorporated herein): ALG2_YEAST, CAPM_STAAU, C69098, C69255,SUS2_MAIZE, A69143, S74778, AB009527_(—)13, AF050103_(—)2 andBBA224769_(—)1.

Example 24 Isolation of cDNA Clones Encoding Human PRO1313 Polypeptides[UNQ679]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), a proprietaryEST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.), andproprietary ESTs from Genentech. The search was performed using thecomputer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap. This consensus sequence is herein designated DNA64876.Based on the DNA64876 consensus sequence and upon a search for sequencehomology with a proprietary Genentech EST sequence designated asDNA57711, a Merck/Washington University EST sequence designated R80613was found to have significant homology with DNA64876 and DNA57711.Therefore, the Merck/Washington University EST clone no. R80613 waspurchased and the insert thereof obtained and sequence, thereby givingrise to the DNA64966-1575 sequence shown in FIG. 41.

DNA sequencing of the R80613 insert obtained as described above gave thefull-length DNA sequence for PRO1313 (designated herein as DNA64966-1575[FIG. 41, SEQ ID NO: 41]; (UNQ679) and the derived protein sequence forPRO1313.

The entire nucleotide sequence of UNQ679 (DNA64966-1575) is shown inFIG. 41 (SEQ ID NO:41). Clone UNQ679 (DNA64966-1575) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 115-117 and ending at the stop codon at nucleotidepositions 1036-1038 (FIG. 41). The predicted polypeptide precursor is307 amino acids long (FIG. 42; SEQ ID NO:42). The full-length PRO1313protein shown in FIG. 42 has an estimated molecular weight of about35,098 daltons and a pI of about 8.11. Analysis of the full-lengthPRO1313 sequence shown in FIG. 42 (SEQ ID NO:42) evidences the presenceof the following: a signal peptide from about amino acid 1 to aboutamino acid 15, transmembrane domains from about amino acid 134 to aboutamino acid 157, from about amino acid 169 to about amino acid 189, fromabout amino acid 230 to about amino acid 248 and from about amino acid272 to about amino acid 285, potential N-glycosylation sites from aboutamino acid 34 to about amino acid 37, from about amino acid 135 to aboutamino acid 138 and from about amino acid 203 to about amino acid 206 andATP/GTP binding site motif A from about amino acid 53 to about aminoacid 60. Clone UNQ679 (DNA64966-1575) has been deposited with ATCC onJan. 12, 1999 and is assigned ATCC deposit no. 203575.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 42 (SEQ ID NO:42), evidenced significant homology betweenthe PRO1313 amino acid sequence and the following Dayhoff sequences:CELT27A1_(—)3, CEF09C6_(—)7, U93688_(—)9, H64896, YDCX_ECOLI andRNU06101_(—)1.

Example 25 Isolation of cDNA Clones Encoding Human PRO1570 Polypeptides[UNQ776]

A consensus DNA sequence encoding PRO1570 was assembled relative toother EST sequences using phrap as described in Example 1 above to forman assembly. This consensus sequence is designated herein as “DNA65415”.Based on the DNA65415 consensus sequence and other discoveries andinformation provided herein, the clone including Incyte EST 3232285(from a uterine/colon cancer tissue library) was purchased and sequencedin full which gave SEQ ID NO:43.

The entire coding sequence of PRO1570 is included in FIG. 43 (SEQ IDNO:43). Clone DNA68885-1678 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 210-212and an apparent stop codon at nucleotide positions 1506-1508 of SEQ IDNO:43. The predicted polypeptide precursor is 432 amino acids long. FIG.44 (SEQ ID NO:44) shows a number of motifs. Clone DNA68885-1678 has beendeposited with the ATCC on Oct. 6, 1998 and is assigned ATCC deposit no.203311. The full-length PRO1570 protein shown in FIG. 44 has anestimated molecular weight of about 47,644 daltons and a pI of about5.18.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 44 (SEQ ID NO:44), revealed sequence identity between thePRO1570 amino acid sequence and the following Dayhoff sequences(incorporated herein): P_W22986, TMS2_HUMAN, HEPS_HUMAN, P_R89435,AB002134_(—)1, KAL_MOUSE, ACRO_HUMAN, GEN12917, AF045649_(—)1, andP_W34285.

Example 26 Isolation of cDNA Clones Encoding Human PRO1886 Polypeptides[UNQ870]

An initial DNA sequence was identified using a yeast screen, in a humanaortic endothelial cDNA library that preferentially represents the 5′ends of the primary cDNA clones. This sequence was compared to ESTs frompublic databases (e.g., GenBank, Merck/Wash U.), and a proprietary ESTdatabase (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.), usingthe computer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)]. The ESTs were clustered and assembledinto a consensus DNA sequence using the computer program “phrap” (PhilGreen, University of Washington, Seattle, Wash.;http://bozeman.mbt.washington.edu/phrap.docs/phrap.html). This consensussequence is designated herein as “DNA78722”. Other novel sequences wereidentified in the alignment of sequences which formed DNA78722. Based onthe DNA78722 consensus sequence, oligonucleotides were synthesized foruse as probes to isolate a clone of the full-length coding sequence forPRO1886 from a human aortic endothelial cells cDNA library.

The full length DNA80796-2523 clone shown in FIG. 45 contained a singleopen reading frame with an apparent translational initiation site atnucleotide positions 73-75 and ending at the stop codon found atnucleotide positions 1022-1025 (FIG. 45; SEQ ID NO:45). The predictedpolypeptide precursor (FIG. 46, SEQ ID NO:46) is 316 amino acids long.Other features are indicated in FIG. 46. PRO1886 has a calculatedmolecular weight of approximately 36045 daltons and an estimated pI ofapproximately 8.18. Clone DNA80796-2523 (UNQ870), designated asDNA80796-2523 has been deposited with the ATCC on Dec. 22, 1998 and isassigned ATCC deposit no. 203555.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 46 (SEQ ID NO:46), revealed sequence identity between thePRO1886 amino acid sequence and the following Dayhoff sequences:CELT26A8_(—)2 and S43230.

Example 27 Isolation of cDNA Clones Encoding Human PRO1891 Polypeptides[UNQ873]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), and aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Aconsensus DNA sequence encoding PRO1891 was assembled relative to otherEST sequences using phrap. This consensus sequence is designated herein“DNA44813”

Based on the DNA44813 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO1891. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primers: GCTGCTTTGCTCACAACTGCTCGC,(44813.f1; SEQ ID NO: 129) CATGACACCTTCCTGCTG (44813.f2; SEQ ID NO: 130)and CAGCCATGGGTGACTGTGACCTCC (44813.f3; SEQ ID NO: 131)reverse PCR primers: CTCCTGGGAGTCGGTAGCAACACC,(44813.r1; SEQ ID NO: 132) GGGAGGTCACAGTCACCC (44813.r2; SEQ ID NO: 133)and GGCTGGGCTTTCCACCCTGGCAC. (44813.r3; SEQ ID NO: 134)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA44813 sequence which had the followingnucleotide sequence:

hybridization probe: (44813.p1; SEQ ID NO: 135)CAGCCATGGGTGACTGTGACCTCCCTGAGTTTTGCACGGG.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1891 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated human bonemarrow. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith olio dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1891, designated herein as“DNA76788-2526”(FIG. 47; SEQ ID NO:47), and the derived protein sequencefor PRO1891.

The entire coding sequence of PRO1891 is shown in FIG. 47 (SEQ IDNO:47). Clone DNA76788-2526 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 114-116,and an apparent stop codon at nucleotide positions 2553-2555. Thepredicted polypeptide precursor is 813 amino acids long. The full-lengthPRO1891 protein shown in FIG. 48 (SEQ ID NO:48) has an estimatedmolecular weight of about 87,739 daltons and a pI of about 6.94.Additional features include a signal peptide at about amino acids 1-27;a transmembrane domain at about amino acids 702-720; potentialN-glycosylation sites at about amino acids 109-112, 145-148, 231-234,276-279, and 448-451; a tyrosine kinase phosphorylation site at aboutamino acids 236-243; potential N-myristoylation sites at about aminoacids 29-34, 285-190, 195-200, 308-313, 318-323, 326-331, 338-343,370-375, 400-405, 402-407, 454-459, 504-509, 510-515, 517-522, 580-585,601-606, 661-666, 687-692, 717-722, and 719-724; an amidation site atabout amino acids 200-203; and a neutral zinc metallopeptidases,zinc-binding region signature at about amino acids 342-351.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 48 (SEQ ID NO:48), revealed significant homology betweenthe PRO1891 amino acid sequence and the following Dayhoff sequences:XLU66003_(—)1, P_W25716, AF023477_(—)1, P_W01825, P_R99801, P_W25722,P_W44120, P_R67759, AF029899_(—)1, and P_W14772.

Clone DNA76788 (UNQ873), designated as DNA76788-2526 was deposited withthe ATCC on Dec. 22, 1998 and is assigned ATCC deposit no. 203551.

Example 28 Isolation of cDNA Clones Encoding Human PRO4409 Polypeptides[UNQ1934]

DNA88004-2575 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the Incyte database. ThisEST cluster sequence was then compared to a variety of expressedsequence tag (EST) databases which included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) to identify existing homologies. Thehomology search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto a consensus DNA sequence with the program “phrap” (Phil Green,University of Washington, Seattle, Wash. The consensus sequence obtainedtherefrom is herein designated DNA79305. In light of DNA79305, a humanbrain library cDNA library was screened with the following two primersto identify DNA88004-2575: 5′GAGCTGAAGTCAGCCTTTGAG3′ (SEQ ID NO:136,forward) and 5′CTCTGCAGAAGTCTCGTTCC3′ (SEQ ID NO:137, reverse).

The full length clone shown in FIG. 49 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 337-339 and ending at the stop codon found at nucleotidepositions 1171-1173 (FIG. 49; SEQ ID NO:49). The predicted polypeptideprecursor (FIG. 50, SEQ ID NO:50) is 278 amino acids long. PRO4409 has acalculated molecular weight of approximately 30748 daltons and anestimated pI of approximately 5.47.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 50 (SEQ ID NO:50), revealed homology between the PRO4409amino acid sequence and the following Dayhoff sequences (sequences nadrelated text incorporated herein): HGS_RF300, HSU80744_(—)1,CEC11H1_(—)7, CEVK04G11_(—)2, HGS_RF177, CEF09E8_(—)2, AF034802_(—)1,P_R51227, I46014 and CYL2_BOVIN.

Clone DNA88004-2575 (UNQ1934), designated as DNA88004-2575 was depositedwith the ATCC on Mar. 30, 1999 and is assigned ATCC deposit no. 203890.

Example 29 Isolation of cDNA Clones Encoding Human PRO5725 Polypeptides[UNQ2446]

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified which showed homology to Neuritin. EST clone no. 3705684 wasthen purchased from LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.and the cDNA insert of that clone was obtained and sequenced inentirety.

The entire nucleotide sequence of the clone, designated herein asDNA92265-2669, is shown in FIG. 51 (SEQ ID NO: 51). The DNA92265-2669clone contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 27-29 and a stopsignal at nucleotide positions 522-524 (FIG. 51, SEQ ID NO:51). Thepredicted polypeptide precursor is 165 amino acids long, has acalculated molecular weight of approximately 17,786 daltons and anestimated pI of approximately 8.43. Analysis of the full-length PRO5725sequence shown in FIG. 52 (SEQ ID NO:52) evidences the presence of avariety of important polypeptide domains as shown in FIG. 52, whereinthe locations given for those important polypeptide domains areapproximate as described above. Clone DNA92265-2669 has been depositedwith ATCC on Jun. 22, 1999 and is assigned ATCC deposit no. PTA-256.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 52 (SEQ ID NO:52), evidenced sequence identity between thePRO5725 amino acid sequence and the following Dayhoff sequences:RNU88958_(—)1; P_W37859; P_W37858; JC6305; HGS_RE778; HGS_RE777;P_W27652; P_W44088; HGS_RE776; and HGS_RE425.

Example 30 Isolation of cDNA Clones Encoding Human PRO6097 Polypeptides[UNQ2545]

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from human SK-Lu-1 adenocarcinoma cell line tissueusing reagents and protocols from Invitrogen, San Diego, Calif. (FastTrack 2). This RNA was used to generate an oligo dT primed cDNA libraryin the vector pRK5D using reagents and protocols from Life Technologies,Gaithersburg, Md. (Super Script Plasmid System). In this procedure, thedouble stranded cDNA was sized to greater than 1000 bp and the SalI/NotIlinkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is acloning vector that has an sp6 transcription initiation site followed byan SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with the amylase sequence willlead to the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176-179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Klentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

(SEQ ID NO: 67) 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′The sequence of reverse oligonucleotide 2 was:

(SEQ ID NO: 68) 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′

PCR was then performed as follows:

a. Denature 92° C., 5 minutes b.  3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c.  3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides disclosed above annealedto the ADH promoter region and the amylase region, respectively, andamplified a 307 bp region from vector pSST-AMY.0 when no insert waspresent. Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

5. Identification of Full-Length Clone

A cDNA sequence isolated in the above screen is herein designatedDNA84712. Probes were then generated from the sequence of the DNA84712molecule and used to screen a human SK-Lu-1 adenocarcinoma cell linelibrary (247) prepared as described in paragraph 1 above. The cloningvector was pRK5B (pRK5B is a precursor of pRK5D that does not containthe SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)), andthe cDNA size cut was less than 2800 bp. The oligonucleotides probeswere synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO6097. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In order to screen severallibraries for a full-length clone, DNA from the libraries was screenedby PCR amplification, as per Ausubel et al., Current Protocols inMolecular Biology, supra, with the PCR primer pair. A positive librarywas then used to isolate clones encoding the gene of interest using theprobe oligonucleotide and one of the primer pairs.

The oligonucleotide probes employed were as follows:

forward PCR primer (SEQ ID NO: 138) 5′-CTGACCGGTCCGCTCATGG-3′reverse PCR primer (SEQ ID NO: 139) 5′-CAGCATGCTTTCCGCGAAGTC-3′hybridization probe (SEQ ID NO: 140)5′-GGCAGGAAGGCCAGGGGTGCTGAGTTCTTCACCTCCTTTTAGAC TG-3′

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 158-160 and a stop signal at nucleotide positions 1727-1729(FIG. 55, SEQ ID NO: 55). The predicted polypeptide precursor is 523amino acids long, has a calculated molecular weight of approximately58,887 daltons and an estimated pI of approximately 9.57. Analysis ofthe full-length PRO6097 sequence shown in FIG. 56 (SEQ ID NO: 56)evidences the presence of a variety of important polypeptide domains asshown in FIG. 56, wherein the locations given for those importantpolypeptide domains are approximate as described above. CloneDNA107701-2711 has been deposited with ATCC on Aug. 3, 1999 and isassigned ATCC Deposit No. PTA-487.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 56 (SEQ ID NO: 56), evidenced sequence identity betweenthe PRO6097 amino acid sequence and the following Dayhoff sequences:YMB8_YEAST; S49759; ATF10N7_(—)5; SPBC405_(—)3; S69718; H69798; D71226;U95370_(—)5; A69780; B69461.

Example 31 Isolation of cDNA Clones Encoding Human PRO7425 Polypeptides[UNQ2966]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included (1) public EST databases (e.g., Merck/WashingtonUniversity), (2) a proprietary EST database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.), (3) a proprietary EST database fromGenentech. The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA86620. In some cases, the DNA86620 consensus sequencederives from an intermediate consensus DNA sequence which was extendedusing repeated cycles of BLAST and phrap to extend that intermediateconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based on the DNA86620 consensus sequence, and in light of an observedsequence homology between the DNA86620 sequence and an EST sequenceencompassed within clone no. 4797137 from the LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif. database, clone no. 4797137 waspurchased and the cDNA insert was obtained and sequenced. It was foundherein that that cDNA insert encoded a full-length protein. The sequenceof this cDNA insert is shown in FIG. 57 and is herein designated asDNA108792-2753.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 3-5 and a stop signal at nucleotide positions 708-710 (FIG.57, SEQ ID NO: 57). The predicted polypeptide precursor is 235 aminoacids long, has a calculated molecular weight of approximately 25989daltons and an estimated pI of approximately 8.32. Analysis of thefull-length PRO7425 sequence shown in FIG. 58 (SEQ ID NO: 58) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 58, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA108792-2753 hasbeen deposited with ATCC on Aug. 31, 1999 and is assigned ATCC DepositNo. PTA-617.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 58 (SEQ ID NO: 58), evidenced sequence identity betweenthe PRO7425 amino acid sequence and the following Dayhoff sequences:P_Y11831; P_Y11619; MYP0_HUMAN; MYP0_MOUSE; HSPMPO2_(—)1; AF087020_(—)1;GEN13751; AF007783_(—)1; P_W14146; XLU43330_(—)1.

Example 32 Isolation of cDNA Clones Encoding Human PRO10102 Polypeptides[UNQ3103]

DNA129542-2808 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the LIFESEQ® (IncytePharmaceuticals, Inc., Palo Alto, Calif.) database, designated herein as166950H1. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA112560.

In light of an observed sequence homology between the DNA112560 sequenceand an EST sequence encompassed within clone no. 166950 from LIFESEQ®(Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) database, clone no.166950 was purchased and the cDNA insert was obtained and sequenced. Itwas found herein that that cDNA insert encoded a full-length protein.The sequence of this cDNA insert is shown in FIG. 59 and is hereindesignated as DNA129542-2808.

Clone DNA129542-2808 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 58-60 andending at the stop codon at nucleotide positions 1786-1788 (FIG. 59; SEQID NO:59). The predicted polypeptide precursor is 576 amino acids long(FIG. 60; SEQ ID NO:60). The full-length PRO10102 protein shown in FIG.60 has an estimated molecular weight of about 62128 daltons and a pI ofabout 7.41. Analysis of the full-length PRO10102 sequence shown in FIG.60 (SEQ ID NO:60) evidences the presence of a variety of importantpolypeptide domains as shown in FIG. 60, wherein the locations given forthose important polypeptide domains are approximate as described above.Clone DNA129542-2808 has been deposited with ATCC on Feb. 23, 2000 andis assigned ATCC deposit no. PTA-1405.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 60 (SEQ ID NO:60), evidenced sequence identity between thePRO10102 amino acid sequence and the following Dayhoff sequences:rTNFSF3L_(—)1, P_Y00771, AC007785_(—)2, AF076483_(—)1, P_W23722,P_W37837, P_Y00770 and AB016605_(—)1. The PRO10102 polypeptide is muchlonger than the sequences with which it shares homology. For example, ithas 378 amino acid residues at the N-terminal end that are nothomologous to rTNFSF3L_(—)1 and P_Y00771.

Example 33 Isolation of cDNA Clones Encoding Human PRO10282 Polypeptides[UNQ3126]

A cDNA clone (DNA148380-2827) encoding a native human PRO10282polypeptide was identified using a yeast screen, in a human cDNA librarythat preferentially represents the 5′ ends of the primary cDNA clones.

Clone DNA148380-2827-1 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 49-51 andending at the stop codon at nucleotide positions 2050-2052 (FIG. 61; SEQID NO:61). The predicted polypeptide precursor is 667 amino acids long(FIG. 62; SEQ ID NO:62). The full-length PRO10282 protein shown in FIG.62 has an estimated molecular weight of about 73502 daltons and a pI ofabout 9.26. Analysis of the full-length PRO10282 sequence shown in FIG.62 (SEQ ID NO:62) evidences the presence of a variety of importantpolypeptide domains as shown in FIG. 62, wherein the locations given forthose important polypeptide domains are approximate as described above.Clone DNA148380-2827 has been deposited with ATCC on Jan. 11, 2000 andis assigned ATCC deposit no. PTA-1181.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 62 (SEQ ID NO:62), evidenced sequence identity between thePRO10282 amino acid sequence and the following Dayhoff sequences:AF062476, P_W88559 and HGS_RE259.

Example 34 Isolation of cDNA Clones Encoding Human PRO779 Polypeptides[UNQ455]

Human fetal heart and human fetal lung lgt10 bacteriophage cDNAlibraries (both purchased from Clontech) were screened by hybridizationwith synthetic oligonucleotide probes based on an EST (GenBank locusW71984), which showed some degree of homology to the intracellulardomain (ICD) of human TNFR1 and CD95. W71984 is a 523 bp EST, which inits −1 reading frame has 27 identities to a 43 amino acid long sequencein the ICD of human TNFR1. The oligonucleotide probes used in thescreening were 27 and 25 bp long, respectively, with the followingsequences:

5′-GGCGCTCTGGTGGCCCTTGCAGAAGCC-3′ (SEQ ID NO: 141)5′-TTCGGCCGAGAAGTTGAGAAATGTC-3′ (SEQ ID NO: 142)

Hybridization was done with a 1:1 mixture of the two probes overnight atroom temperature in buffer containing 20% formamide, 5×SSC, 10% dextransulfate, 0.1% NaPiPO4) 0.05 M NaPO4, 0.05 mg salmon sperm DNA, and 0.1%sodium dodecyl sulfate (SDS), followed consecutively by one wash at roomtemperature in 6×SSC, two washes at 37 C in 1×SSC/0.1% SDS, two washesat 37 C in 0.5×SSC/0.1% SDS, and two washes at 37 C in 0.2×SSC/0.1% SDS.One positive clone from each of the fetal heart (FH20A.57) and fetallung (FL8A.53) libraries were confirmed to be specific by PCR using therespective above hybridization probes as primers. Single phage plaquescontaining each of the positive clones were isolated by limitingdilution and the DNA was purified using a Wizard lambda prep DNApurification kit (Promega).

The cDNA inserts were excised from the lambda vector arms by digestionwith EcoRI, gel-purified, and subcloned into pRK5 that was predigestedwith EcoRI. The clones were then sequenced in entirety.

Clone (FH20A.57) DNA58801-1052 (also referred to as Apo 3 clone FH20.57deposited as ATCC 55820, as indicated below) contains a single openreading frame with an apparent translational initiation site atnucleotide positions 103-105 and ending at the stop codon found atnucleotide positions 1354-1356 [FIG. 65, SEQ ID NO:65]. The predictedpolypeptide precursor is 417 amino acids long (FIG. 66; SEQ ID NO:66).The full-length PRO779 protein shown in FIG. 66 has an estimatedmolecular weight of about 45,000 daltons and a pI of about 6.40.Analysis of the full-length PRO779 sequence shown in FIG. 66 (SEQ IDNO:66) evidences the presence of a variety of important polypeptidedomains, wherein the locations given for those important polypeptidedomains are approximate as described above. Analysis of the full-lengthPRO779 sequence shown in FIG. 66 evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid24; a transmembrane domain from about amino acid 199 to about amino acid219; N-glycosylation sites from about amino acid 67 to about amino acid71 and from about amino acid 106 to about amino acid 110; a cAMP- andcGMP-dependent protein kinase phosphorylation site from about amino acid157 to about amino acid 161; a tyrosine kinase phosphorylation site fromabout amino acid 370 to about amino acid 377; N-myristoylation sitesfrom about amino acid 44 to about amino acid 50, from about amino acid50 to about amino acid 56, from about amino acid 66 to about amino acid72, from about amino acid 116 to about amino acid 122, from about aminoacid 217 to about amino acid 223, from about amino acid 355 to aboutamino acid 361, from about amino acid 391 to about amino acid 397, andfrom about amino acid 401 to about amino acid 407; and a prokaryoticmembrane lipoprotein lipid attachment site from about amino acid 177 toabout amino acid 188. Clone DNA58801-1052 has been deposited with ATCCon Sep. 5, 1996 and is assigned ATCC deposit no. 55820.

The ECD contains 4 cysteine-rich repeats which resemble thecorresponding regions of human TNFR1 (4 repeats), of human CD95 (3repeats) and of the other known TNFR family members. The ICD contains adeath domain sequence that resembles the death domains found in the ICDof TNFR1 and CD95 and in the cytoplasmic death signaling proteins suchas human FADD/MORT1, TRADD, RIP, and Drosophila Reaper. Both globallyand in individual regions, PRO779 (Apo 3) is more closely related toTNFR1 than to CD95; the respective amino acid identities are 29.3% and22.8% overall, 28.2% and 24.7% in the ECD, 31.6% and 18.3% in the ICD,and 47.5% and 20% in the death domain.

Example 35 Generation and Analysis of Mice Comprising PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 GeneDisruptions

To investigate the role of PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides, disruptions in PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 geneswere produced by homologous recombination or retroviral insertiontechniques. Specifically, transgenic mice comprising disruptions inPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 genes (i.e., knockout mice) were created by either gene targetingor gene trapping. Mutations were confirmed by southern blot analysis toconfirm correct targeting on both the 5′ and 3′ ends. Gene-specificgenotyping was also performed by genomic PCR to confirm the loss of theendogenous native transcript as demonstrated by RT-PCR using primersthat anneal to exons flanking the site of insertion. Targeting vectorswere electroporated into 129 strain ES cells and targeted clones wereidentified. Targeted clones were microinjected into host blastocysts toproduce chimeras. Chimeras were bred with C57 animals to produce F1heterozygotes. Heterozygotes were intercrossed to produce F2 wild-type,heterozygote and homozygote cohorts which were used for phenotypicanalysis. Rarely, if not enough F1 heterozygotes were produced, the F1hets were bred to wild-type C57 mice to produce sufficient heterozygotesto breed for cohorts to be analyzed for a phenotype. All phenotypicanalysis was performed from 12-16 weeks after birth.

Overall Summary of Results

35.1. Generation and Analysis of Mice Comprising DNA22779-1130 (UNQ170)Gene Disruptions

In these knockout experiments, the gene encoding PRO196 polypeptides(designated as DNA22779-1130) (UNQ170) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)011923 or ACCESSION:NM_(—)011923 NID: gi 6753119 ref NM_(—)011923.1 Mus musculusangiopoietin-like 2 (Angptl2); protein reference: Q9R045 or ACCESSION:Q9R045NID: Mus musculus (Mouse). ANGIOPOIETIN-RELATED PROTEIN 2PRECURSOR (ANGIOPOIETIN-LIKE 2). MOUSESPTRNRDB; the human gene sequencereference: NM_(—)012098 or ACCESSION: NM_(—)012098 NID: gi 6912235 refNM_(—)012098.1 Homo sapiens angiopoietin-like 2 (ANGPTL2); the humanprotein sequence corresponds to reference: Q9UKU9 or ACCESSION: Q9UKU9NID: Homo sapiens (Human). ANGIOPOIETIN-RELATED PROTEIN 2 PRECURSOR(ANGIOPOIETIN-LIKE 2). HUMANSPTRNRDB.

The mouse gene of interest is angiopoietin-like 2 (Angptl2), ortholog ofhuman ANGPTL2. Aliases include angiopoietin related protein 2 (Arp2),HARP, and MGC8889. Angptl2 is a secreted glycoprotein hormone expressedin vascular endothelial cells, vascular smooth muscle, heart, smallintestine, spleen, stomach, colon, ovary, adrenal gland, skeletalmuscle, and prostate. Although Angptl2 is structurally similar to theangiopoietin family of hormones, Angptl2 does not bind to angiopoietinreceptors Tie1 and Tie2. Angptl2 induces sprouting in endothelial cells,which is consistent with the role of angiopoietins in blood vesselformation (Kim et al., J Biol Chem, 274(37):26523-8 (1999)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 23 32 23 78 Expected 19.5 39 19.5 78 Chi-Sq. =2.51 Significance = 0.28467 (hom/n) = 0.29 Avg. Litter Size = 8Mutation Type: Homologous Recombination (standard).Coding exon 1 was targeted (NCBI accession NM_(—)011923.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptskeletal muscle, bone, and adipose.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.1.1. Phenotypic Analysis (for Disrupted Gene: DNA22779-1130 (UNQ170)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human angiopoietin-like 2(ANGPTL2) resulted in decreased mean serum MCP-1, TNF-alpha, and IL-6responses to LPS challenge in (−/−) mice. The (−/−) mice also exhibitedsignificant growth retardation marked by decreased total tissue mass,total body fat, decreased body weight and length as well as decreasedmean vertebral trabecular bone measurements. Gene disruption wasconfirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACSCalibur instrument.

Results:

The (−/−) mice exhibited a decreased mean serum MCP-1, IL-6 andTNF-alpha response to LPS challenge when compared with their (+/+)littermates and the historical mean.

Analyzed wt/het/hom: 6/4/10

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO196 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited a decreased ability to elicit animmunological response (MCP-1, TNF-alpha and IL-6 production) whenchallenged with the LPS endotoxin indicating a deficiency in theproinflammatory response. IL-6 contributes to the later stages of B cellactivation. In addition, IL-6 plays a critical role in inducing theacute phase response and systemic inflammation. This suggests thatPRO196 polypeptides or agonists thereof would stimulate the immunesystem and would find utility in the cases wherein this effect would bebeneficial to the individual such as in the case of leukemia, and othertypes of cancer, and in immunocompromised patients, such as AIDSsufferers. Accordingly, inhibitors or antagonists to PRO196 polypeptideswould play a role in inhibiting the immune response and would be usefulcandidates for suppressing harmful immune responses, e.g. in the case ofgraft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited decreased mean body weight and mean body length(at least two (2) standard deviations (SD) below normal especially inthe first eight (8) weeks) when compared with their gender-matched (+/+)littermates and the historical means.

Analyzed wt/het/hom: 23/32/23

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean total tissue mass andtotal body fat when compared with their gender-matched (+/+) littermatesand the historical means.

Micro-CT: The (−/−) mice exhibited a decreased mean vertebral trabecularbone volume, thickness, and connectivity density when compared withtheir gender-matched (+/+) littermates and the historical means.

Analyzed wt/het/hom: 4/4/10

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO196 polypeptides or agonists thereof play a role in maintaining bonehomeostasis. In addition, PRO196 or its encoding gene would be useful inbone healing or useful for the treatment of osteoarthritis orosteoporosis; whereas antagonists to PRO196 or its encoding gene wouldlead to abnormal or pathological bone disorders including inflammatorydiseases associated with abnormal bone metabolism such as arthritis,osteoporosis, and osteopenia.

The (−/−) mice analyzed by DEXA exhibited significant growth retardationmarked by a notably decreased total tissue mass and total body fat aswell as reduction in body weight and length when compared with their(+/+) littermates. These results may be due to problems associated withthe hypothalamic-pituitary axis which can affect bone growth. This inconjunction with the observations of decreased bone measurements suggesta tissue wasting condition such as cachexia. Thus, PRO196 polypeptidesor agonists thereof would be useful in the treatment of bone disordersbut would also be useful for treating growth disorders or for theprevention of cachexia or other tissue wasting diseases.

35.2. Generation and Analysis of Mice Comprising DNA33094-1131 (UNQ191)Gene Disruptions

In these knockout experiments, the gene encoding PRO217 polypeptides(designated as DNA33094-1131) (UNQ191) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)011915 or ACCESSION:NM_(—)011915 NID: 6755998 Mus musculus Mus musculus Wnt inhibitoryfactor 1 (Wif1); protein reference: Q9WUA1 or ACCESSION: Q9WUA1 NID: Musmusculus (Mouse). WNT INHIBITORY FACTOR 1 PRECURSOR (WIF-1).MOUSESPTRNRDB; the human gene sequence reference: NM_(—)007191 orACCESSION: NM_(—)007191 NID: 18379354 Homo sapiens Homo sapiens WNTinhibitory factor 1 (WIF1); the human protein sequence corresponds toreference: Q9Y5W5 or ACCESSION: Q9Y5W5 NID: Homo sapiens (Human). Wntinhibitory factor 1 precursor (WIF-1). HUMANSPTRNRDB.

The mouse gene of interest is Wif1 (Wnt inhibitory factor 1), orthologof human WIF1. Aliases include WIF-1 and Wnt inhibitory factor-1. WIF1is a secreted protein expressed during embryonic development that bindswith Wnt proteins, disrupting activation of their cognate receptors. Wntproteins are extracellular signaling molecules involved in developmentalprocesses (Hseih et al., Nature, 398(6726):431-6 (1999). WIF1 isdown-regulated in prostate, breast, lung, and bladder cancer butup-regulated in colon adenocarcinoma cell lines, suggesting thatalterations in WIF1 expression may be related to tumorigenesis in thesetissues (Wissmann et al.,

J Pathol, 201(2):204-12 (2003); Cebrat et al., Cancer Lett,206(1):107-13 (2004)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 24 35 20 79 Expected 19.75 39.5 19.75 79Chi-Sq. = 1.43 Significance = 0.48910 (hom/n) = 0.25 Avg. Litter Size =8Mutation Type: Homologous Recombination (standard)Coding exon 1 was targeted (NCBI accession NM_(—)011915.1).Wild-type expression of the target gene was detected in brain, spinalcord, eye, and heart among the 13 adult tissue samples tested by RT-PCR.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.2.1. Phenotypic Analysis (for Disrupted Gene: DNA33094-1131 (UNQ191)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human Wnt inhibitoryfactor 1 (WIF1) resulted in growth retardation accompanied by decreasedbone measurements in (−/−) mice. In addition, mutant (−/−) miceexhibited an increased IgG2a response to an ovalbumin challenge. Genedisruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmuno-dominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Analyzed wt/het/hom: 8/4/9

Results of this Challenge:

The (−/−) mice exhibited an increased mean serum IgG2a response toovalbumin challenge when compared with their (+/+) littermates and thehistorical means. Thus, these knockout mice exhibited an increasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen or a positive immunological phenotype(proinflammatory response). Thus, the gene encoding PRO217 polypeptideswould be expected to lead to inhibition of the proinflammatory responsewhich could be caused by Th, B or plasma cell defects.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO217 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited an increased ability to elicit animmunological response when challenged with the T-cell dependent OVAantigen. Thus, inhibitors or antagonists of PRO217 polypeptides would beuseful for stimulating the immune system (such as T cell proliferation)and would find utility in the cases wherein this effect would bebeneficial to the individual such as in the case of leukemia, and othertypes of cancer, and in immunocompromised patients, such as AIDSsufferers. Accordingly, PRO217 polypeptides or agonists thereof would beuseful for inhibiting the immune response and thus would be usefulcandidates for suppressing harmful immune responses, e.g. in the case ofgraft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited decreased mean body weight and mean body length(one (1) standard deviation (SD) below historic mean in weight and two(2) standard deviations (SD) below historical mean in length) whencompared with their gender-matched (+/+) littermates and the historicalmeans.

Heart Rate: The heart rate decreased 1-2 standard deviations relative tothe historic controls in both male and female knockout (−/−) mice.

Fertility: The single male (−/−) mouse (M-151) available for analysiswas infertile.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

The (−/−) mice exhibited decreased mean total tissue mass and lean bodymass and decreased mean bone mineral content and bone mineral contentwhen compared with their gender-matched (+/+) littermates and thehistorical means. Analyzed wt/het/hom: 4/4/8

Summary

The (−/−) mice analyzed by DEXA exhibited notably decreased total tissuemass and lean body mass as well as decreased bone measurements whencompared with their (+/+) littermates, suggestive of growth retardationin these mutants. This in conjunction with the observations of decreasedbody weight and length suggest a tissue wasting condition such ascachexia or other growth related disorders. Thus, PRO217 polypeptides oragonists thereof would be useful in the treatment or prevention ofgrowth disorders including cachexia or other tissue wasting diseases.

35.3. Generation and Analysis of Mice Comprising DNA34434-1139 (UNQ205)Gene Disruptions

In these knockout experiments, the gene encoding PRO231 polypeptides(designated as DNA34434-1139) (UNQ205) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019800 or ACCESSION:NM_(—)019800 NID: 9790058 Mus musculus Mus musculus acid phosphatase 6,lysophosphatidic (Acp6); protein reference: Q9JMG5 or ACCESSION: Q9JMG5NID: Mus musculus (Mouse). MPACPL1. MOUSESPTRNRDB; the human genesequence reference: NM_(—)016361 or ACCESSION: NM_(—)016361 NID:21359910 Homo sapiens Homo sapiens LPAP for lysophosphatidic acidphosphatase (LOC51205); the human protein sequence corresponds toreference: Q9NPH0 or ACCESSION: Q9NPH0 NID: Homo sapiens (Human).HPACPL1 (CDNA FLJ20650 FIS, CLONE KAT01962) (LPAP FOR LYSOPHOSPHATIDICACID PHOSPHATASE) (LYSOPHOSPHATIDIC ACID PHOSPHATASE PRECURSOR).HUMANSPTRNRDB.

The targeted mouse gene is Acp6 (acid phosphatase 6, lysophosphatidic),which is the ortholog of human ACP6. Aliases include: ACPL1; mPACPL1;5730559A09Rik; acid phosphatase like 1; LPAP; PACPL1; and likelyortholog of mouse acid phosphatase 6, lysophosphatidic. ACP6 is acytoplasmic or mitochondrial enzyme that catalyzes the hydrolysis oflysophosphatidic acid to monoacylglycerol and phosphate. Expression ofACP6 is detected in a wide variety of tissues but is particularly highin kidney, heart, small intestine, muscle, and liver. The enzyme alsohas been detected in the cytosolic fraction of brain homogenates and ininterstitial cells of Cajal (ICC), which function as pacemakers andmediators of motor neurotransmission in gastrointestinal smooth muscle.ACP6 may play a role in lipid metabolism (Hiroyama and Takenawa, BiochemJ, 336 (Pt 2): 483-9 (1998); Hiroyama and Takenawa, J Biol Chem, 274(41): 29172-80 (1999); Takayama et al., Gut 50(6):790-6 (2002)).

Genetics Information:

wt het hom Total Observed 19 39 23 81 Expected 20.25 40.5 20.25 81Chi-Sq. = 0.51 Significance = 0.77640 (hom/n) = 0.28 Avg. Litter Size =8

Mutation Type: Retroviral Insertion (OST).

Retroviral insertion occurred in the intron between coding exons 1 and 2(Accession: NM_(—)019800.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except skeletal muscle, bone, andadipose.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-46). Disruption of the target gene was confirmed byInverse PCR.

35.3.1. Phenotypic Analysis (for Disrupted Gene: DNA34434-1139 (UNQ205)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human acid phosphatase 6,lysophosphatidic (ACP6) resulted in the observation of an increased meanplatelet count in the (−/−) mutant mice compared with the wild-type(+/+) littermates. In addition, the (−/−) mutant mice appeared to havedecreased total body fat (% and gram) as well as decreased total tissuemass indicative of tissue wasting diseases. Transcript was absent byRT-PCR.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

The (−/−) mice exhibited an increased mean platelet count when comparedwith their (+/+) littermates and the historical mean. Analyzedwt/het/hom: 7/5/8

Thus, mutant mice deficient in the DNA34434-1139 gene resulted in aphenotype related to coagulation disorders. In this regard, inhibitorsor antagonists of PRO231 polypeptides would be useful in treatingdisorders related to abnormal blood coagulation such as hemophilia.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The (−/−) mice exhibited a decreased total body fat (% and g) andtotal tissue mass when compared with their gender-matched (+/+)littermates and the historical means.

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbody measurements with significant decreased body fat and masssuggestive of tissue wasting diseases. The (−/−) mice analyzed by DEXAexhibited notably decreased total tissue mass and lean body mass whencompared with their (+/+) littermates, suggestive of growth retardationin these mutants. Thus, PRO231 polypeptides or agonists thereof would beuseful for treating growth disorders or for the prevention of cachexiaor other tissue wasting diseases.

35.4. Generation and Analysis of Mice Comprising DNA35599-1168 (UNQ210)Gene Disruptions

In these knockout experiments, the gene encoding PRO236 polypeptides(designated as DNA35599-1168) (UNQ210) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)153803 or ACCESSION:NM_(—)153803 NID: gi 24418924 ref NM_(—)153803.1 Mus musculushypothetical protein MGC47419 (MGC47419); protein reference: Q8CFT1 orACCESSION: Q8CFT1 NID: Mus musculus (Mouse) Similar to RIKEN cDNA4921509F24 gene; the human gene sequence reference: NM_(—)138342 orACCESSION: NM_(—)138342 NID: gi 24308391 ref NM_(—)138342.1 Homo sapienshypothetical protein BC008326 (LOC89944); the human protein sequencecorresponds to reference: Q81W92 or ACCESSION: Q81W92 NID: Homo sapiens(Human). Hypothetical protein BC008326.

The mouse gene of interest is “cDNA sequence BC038479” (BC038479),ortholog of human “hypothetical protein BC008326.” Aliases includehypothetical protein MGC47419.

BC038479 is a putative lysosomal enzyme that contains a glycosylhydrolase family 35 domain. Proteins with this domain includebeta-galactosidase, a lysosomal enzyme that catalyzes the cleavage ofthe terminal galactose from gangliosides, glycoproteins, andglycosaminoglycans (Pfam accession PF01301).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 22 25 26 73 Expected 18.25 36.5 18.25 73Chi-Sq. = 7.68 Significance = 0.02144 (hom/n) = 0.36 Avg. Litter Size =7Mutation Type: Homologous Recombination (standard)Coding exon 1 was targeted (NCBI accession NM_(—)153803.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.4.1. Phenotypic Analysis (for Disrupted Gene: DNA35599-1168 (UNQ210)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a hypothetical humanprotein (BC008326) resulted in decreased mean white blood cell (WBC) andlymphocyte counts and a decreased mean percentage of natural killercells in (−/−) mice. The (−/−) mice also exhibited impaired glucosetolerance. In addition, the (−/−) mutant mice exhibited abnormal bonemeasurements indicative of osteoporosis. Gene disruption was confirmedby Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

(1) Flourescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

(2) Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

The (−/−) mice exhibited decreased mean total white blood cell andabsolute lymphocyte counts when compared with their (+/+) littermatesand the historical means.

FACS analysis also demonstrated that (−/−) mice exhibited a decreasedmean percentage of natural killer cells when compared with their (+/+)littermates and the historical mean.

Analyzed wt/het/hom: 7/5/8

In summary, the hematology and FACS results indicate that the homozygousmutant mice have an impaired immune system, especially in view of thedecreased total white blood cell count and absolute lymphocyte counts.Furthermore, the decreased mean percentage of natural killer cells is anadditional indicator of a negative phenotype associated with knockingout the DNA35599-1168 gene which encodes PRO236 polypeptides. Naturalkiller cells are the first line of defense to viral infection sincethese cells have been implicated in viral immunity and in defenseagainst tumors. Natural killer cells or NK cells act as effectors inantibody-dependent cell-mediated cytotoxicity and have been identifiedby their ability to kill certain lymphoid tumor cell lines in vitrowithout the need for prior immunization or activation. However, theirknown function in host defense is in the early phases of infection withseveral intracellular pathogens, particularly herpes viruses. Thus,PRO236 polypeptides and agonists thereof would be important for ahealthy immune system and would be useful in stimulating the immunesystem especially against viral infections.

(c) Bone Metabolism & Body Diagnostics: Bone Metabolism: RadiologyPhenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro-CT: The (−/−) mice exhibited decreased bone measurements in the5^(th) lumbar vertebrae (decreased mean vertebral trabecular bone volumeand thickness) when compared with their gender-matched (+/+) littermatesand the historical means.

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO236 polypeptides or agonists thereof play a role in maintaining bonehomeostasis. In addition, PRO236 or its encoding gene would be useful inbone healing or useful for the treatment of arthritis or osteoporosis;whereas antagonists to PRO236 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism such as arthritis,osteoporosis, and osteopenia.

(d) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygote mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/hom: 4/4/8

Results:

During the glucose tolerance test the (−/−) mice exhibited impairedglucose tolerance when compared with their gender-matched (+/+)littermates and the historical means.

These studies indicated that (−/−) mice exhibit a decreased glucosetolerance in the presence of normal fasting glucose at all 3 intervalstested when compared with their gender-matched (+/+) littermates and thehistorical means. Thus, knockout mice exhibited the phenotypic patternof an impaired glucose homeostasis. In light of this observation, PRO236polypeptides (or agonists thereof) or its encoding gene would play beuseful in the treatment of impaired glucose homeostasis and/or variouscardiovascular diseases, including the treatment of diabetes.

35.5. Generation and Analysis of Mice Comprising DNA35638-1141 (UNQ219)Gene Disruptions

In these knockout experiments, the gene encoding PRO245 polypeptides(designated as DNA35638-1141) (UNQ219) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)023844 or Mus musculus RIKENcDNA 1110002N23 gene (1110002N23Rik); protein reference: Q9JI59 orVASCULAR ENDOTHELIAL JUNCTION-ASSOCIATED MOLECULE (JUNCTIONAL ADHESIONMOLECULE-3) (2410030G21RIK PROTEIN); the human gene sequence reference:NM_(—)021219 or ACCESSION: NM_(—)021219 NID: gi 21704284 refNM_(—)021219.2 Homo sapiens junctional adhesion molecule 2 (JAM2); thehuman protein sequence corresponds to reference: P57087 or ACCESSION:P57087 NID: Homo sapiens (Human). JUNCTIONAL ADHESION MOLECULE 2PRECURSOR (VASCULAR ENDOTHELIAL JUNCTION-ASSOCIATED MOLECULE) (VE-JAM)HUMANSPTRNRDB.

The disrupted mouse gene is Jam2 (junction adhesion molecule 2), theortholog of human JAM2. Aliases include VEJAM, VE-JAM, JAM-2, Jcam2,C21orf43, chromosome 21 open reading frame 43, vascular endothelialjunction-associated molecule, and junction cell adhesion molecule 2.

JAM2, an Ig superfamily member, is a membrane-lodged extracellularprotein. JAM2 is specifically localized at junctions of lymphaticendothelial cells and high endothelial venules within lymph nodes andPeyer patches (Johnson-Leger et al., Blood, 100(7):2479-86 (2002)). JAM2binds T, NK, and dendritic cells via interactions with alpha4-beta1integrin and JAM3 (Liang et al., J Immunol, 168(4):1618-26 (2002)). JAM2apparently plays a central role in tight junction formation,transendothelial and lymphocyte migration, and the establishment of cellpolarity in endothelial tissue (Ebnet et al., J Cell Sci 116(Pt19):3879-91 (2003), Aurrand-Lions et al., Cells Tissues Organs,172(3):152-60 (2002)).

Genetics Information:

wt het hom Total Observed 18 32 22 72 Expected 18 36 18 72 Chi-Sq. =1.33 Significance = 0.51342 (hom/n) = 0.31 Avg. Litter Size = 7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred between coding exons 3 and 4 (Accession:NM_(—)023844).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptadipose.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (F-58).

35.5.1. Phenotypic Analysis (for Disrupted Gene: DNA35638-1141 (UNQ219)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human junction adhesionmolecule 2 (JAM2) resulted in an increased anxiety-related response in(−/−) mice. In addition, mutant (−/−) mice demonstrated a decreasedovalbumin response compared with their (+/+) wild-type littermates.Also, the (−/−) mutant mice showed opthamological abnormalitiesassociated with an increase in retinal artery tortuosity. Transcript wasabsent by RT-PCR.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmuno-dominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Analyzed wt/het/hom: 8/4/9

Results of this Challenge:

The (−/−) mice exhibited a decreased mean serum IgG2a response toovalbumin challenge when compared with their (+/+) littermates and thehistorical means. Thus, these knockout mice exhibited a decreasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO245 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited a decreased ability to elicit animmunological response when challenged with the T-cell dependent OVAantigen. Thus, PRO245 polypeptides or agonists thereof, would be usefulfor stimulating the immune system (such as T cell proliferation) andwould find utility in the cases wherein this effect would be beneficialto the individual such as in the case of leukemia, and other types ofcancer, and in immunocompromised patients, such as AIDS sufferers.Accordingly, inhibitors (antagonists) of PRO245 polypeptides would beuseful for inhibiting the immune response and thus would be usefulcandidates for suppressing harmful immune responses, e.g. in the case ofgraft rejection or graft-versus-host diseases.

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value. Analyzed wt/het/hom: 5/4/8

Results:

The (−/−) mice exhibited a decreased median sum time-in-center duringopen field testing when compared with their gender-matched (+/+)littermates and the historical mean, suggesting an increasedanxiety-like response in the mutants. As noted above, a notabledifference was observed during open field activity testing. The (−/−)mice exhibited a decreased median sum time in the center area whencompared with their gender-matched (+/+) littermates. This type ofbehavior is consistent with an increased anxiety like response. Thus,the knockout mice demonstrated a phenotype consistent with anxietyrelated disorders which are associated with mild to moderate anxiety,anxiety due to a general medical condition, and/or bipolar disorders;hyperactivity; sensory disorders; obsessive-compulsive disorders,schizophrenia or a paranoid personality. Thus, PRO245 polypeptides oragonists thereof would be useful in the treatment of such neurologicaldisorders or the amelioration of the symptoms associated with anxietydisorders.

(d) Cardiovascular Phenotypic Analysis:

In the area of cardiovascular biology, phenotypic testing was performedto identify potential targets for the treatment of cardiovascular,endothelial or angiogenic disorders. One such phenotypic test includedoptic fundus photography and angiography to determine the retinalarteriovenous ratio (A/V ratio) in order to flag various eyeabnormalities. An abnormal A/V ratio signals such systemic diseases ordisorders that may be related to the vascular disease of hypertension(and any disease that causes hypertension, e.g. atherosclerosis),diabetes or other ocular diseases corresponding to ophthalmologicaldisorders. Such eye abnormalities may include but are not limited to thefollowing: retinal abnormality is retinal dysplasia, variousretinopathies, restenosis, retinal artery obstruction or occlusion;retinal degeneration causing secondary atrophy of the retinalvasculature, retinitis pigmentosa, macular dystrophies, Stargardt'sdisease, congenital stationary night blindness, choroideremia, gyrateatrophy, Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Optic fundus photography was performed onconscious animals using a Kowa Genesis small animal fundus cameramodified according to Hawes and coauthors (Hawes et al., 1999 MolecularVision 1999; 5:22). Intra-peritoneal injection of fluorescein permittedthe acquisition of direct light fundus images and fluorescent angiogramsfor each examination. In addition to direct ophthalmological changes,this test can detect retinal changes associated with systemic diseasessuch as diabetes and atherosclerosis or other retinal abnormalities.Pictures were provided of the optic fundus under normal light. Theangiographic pictures allowed examination of the arteries and veins ofthe eye. In addition an artery to vein (A/V) ratio was determined forthe eye.

Ophthalmology analysis was performed on generated F2 wild type,heterozygous, and homozygous mutant progeny using the protocol describedabove. Specifically, the A/V ratio was measured and calculated accordingto the fundus images with Kowa COMIT+ software. This test takes colorphotographs through a dilated pupil: the images help in detecting andclassifying many diseases. The artery to vein ratio (A/V) is the ratioof the artery diameter to the vein diameter (measured before thebifurcation of the vessels). Many diseases will influence the ratio,i.e., diabetes, cardiovascular disorders, papilledema, optic atrophy orother eye abnormalities such as retinal degeneration (known as retinitispigmentosa) or retinal dysplasia, vision problems or blindness. Thus,phenotypic observations which result in an increased artery-to-veinratio in homozygous (−/−) and heterozygous (+/−) mutant progeny comparedto wild-type (+/+) littermates would be indicative of such pathologicalconditions.

Results:

Pathological microscopic observations showed an increase in retinalartery tortuosity in the (−/−) mice analyzed. In addition, cornealinflammation and ulceration was also noted in the (−/−) mutant mice.Analyzed wt/het/hom: 0/1/4

In summary, in this study, (−/−) mice showed opthamologicalabnormalities which would lead to attenuated retinal vessels andpossibly retinal degeneration when compared with their (+/+)littermates. In summary, by knocking out the gene identified asDNA35638-1141 encoding PRO245 polypeptides, homozygous mutant progenyexhibit phenotypes which are associated with retinal arteryabnormalities. Such detected retinal changes are most commonlyassociated with cardiovascular systemic diseases or disorders that maybe related to the vascular disease of hypertension (and any disease thatcauses hypertension, e.g. atherosclerosis), diabetes or other oculardiseases corresponding to ophthalmological disorders such as retinaldegeneration. Thus, antagonists of PRO245 encoding genes would lead tosimilar pathological retinal changes, whereas agonists would be usefulas therapeutic agents in the treatment of hypertension, atherosclerosisor other opthamological disorders including retinal degeneration anddiseases associated with this condition (as indicated above).

35.6. Generation and Analysis of Mice Comprising DNA35639-1172 (UNQ220)Gene Disruptions

In these knockout experiments, the gene encoding PRO246 polypeptides(designated as DNA35639-1172) (UNQ220) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)027102 OR ACCESSION:NM_(—)027102 NID: 22094994 Mus musculus Mus musculus endothelialcell-selective adhesion molecule; protein reference: Q925F2 orACCESSION: Q925F2 NID: Mus musculus (Mouse). ENDOTHELIAL CELL-SELECTIVEADHESION MOLECULE. MOUSESPTRNRDB; the human gene sequence reference:NM_(—)138961 or ACCESSION: NM_(—)138961 NID: 20452463 Homo sapiens Homosapiens similar to endothelial cell-selective adhesion molecule; thehuman protein sequence corresponds to reference: Q96AP7 or ACCESSION:Q96AP7 NID: Homo sapiens (Human). Hypothetical protein PLACE1000456.

The disrupted mouse gene is Esam1 (endothelial cell-specific adhesionmolecule), ortholog of human ESAM. Aliases include W117m, 2310008D05Rik,and HUEL (C4orf1)-interacting protein.

ESAM is a cell adhesion molecule of the immunoglobulin receptor familyexpressed at tight junctions on endothelial cells. ESAM is likely toplay a role in inter endothelial cell adhesion (Hirata et al., J BiolChem, 276(19):16223-31 (2001); Nasdala et al, J Biol Chem,277(18):16294-303 (2002)). Ishida and colleagues, [J Biol Chem,278(36):34598-604 (2003)] showed that tumor volume of ESAM homozygousnull mice was notably smaller than that of wild-type mice. By matrigelplug assay, Ishida and colleagues also showed that neovascularizationwas notably lower in ESAM homozygous null mice than in wild-type mice.They concluded that ESAM played a role in pathological angiogenicprocesses such as tumor growth.

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 18 26 15 59 Expected 14.75 29.5 14.75 59Chi-Sq. = 1.14 Significance = 0.56677 (hom/n) = 0.25 Avg. Litter Size =7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 2 and 3(accession: NM_(—)027102.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptadipose.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-156). Disruption of the target gene was confirmed byInverse PCR.

35.6.1. Phenotypic Analysis (for Disrupted gene: DNA35639-1172 (UNQ220)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human endothelialcell-specific adhesion molecule (ESAM) resulted in an increased meanserum glucose levels and a decreased or impaired glucose tolerance. Inaddition, (−/−) mice exhibited a decreased heart rate (one standarddeviation below the historic mean). Transcript was absent by RT-PCR.

(b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection.

Results:

The (−/−) mice exhibited an increased mean serum glucose level [two (2)standard deviations (SD) above the historic mean] when compared withtheir gender-matched (+/+) littermates and the historical mean. Inaddition, the (−/−) mice exhibited impaired glucose tolerance whencompared with their gender-matched (+/+) littermates and the historicalmeans.

Analyzed wt/het/hom: 4/4/8

These studies indicated that (−/−) mice exhibit a decreased or impairedglucose tolerance in the presence of normal fasting glucose at all 3intervals tested when compared with their gender-matched (+/+)littermates and the historical means. Thus, knockout mutant miceexhibited the phenotypic pattern of an impaired glucose homeostasis, andtherefor PRO246 polypeptides (or agonists thereof) or its encoding genewould be useful in the treatment of conditions associated with animpaired glucose homeostasis and/or various cardiovascular diseases,including diabetes.

35.7. Generation and Analysis of Mice Comprising DNA35918-1174 (UNQ225)Gene Disruptions

In these knockout experiments, the gene encoding PRO258 polypeptides(designated as DNA35918-1174) (UNQ225) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)053199 or Mus musculusimmunoglobulin superfamily, member 4B (Igsf4b); protein reference:Q99N28 or ACCESSION: Q99N28 NID: Mus musculus (Mouse). NECTIN-LIKEPROTEIN 1. MOUSESPTRNRDB; the human gene sequence reference:NM_(—)021189 or Homo sapiens immunoglobulin superfamily, member 4B(IGSF4B); the human protein sequence corresponds to reference: Q9UJP1 orACCESSION: Q9UJP1 NID: Homo sapiens (Human). BK134P22.1 (NOVEL PROTEINSIMILAR TO MOUSE IMMUNOSUPERFAMILY PROTEIN BL2) (NECTIN-LIKE PROTEIN 1).HUMANSPTRNRDB.

The mouse gene of interest is Igsf4b (immunoglobulin superfamily, member4B), ortholog of human IGSF4B. Aliases include BIgR, Necl1, Tsll1,FLJ10698, nectin-like 1, TSLC1-like 1, nectin-like protein 1, and brainimmunoglobulin receptor precursor.

IGSF4B is a type I plasma membrane protein expressed primarily inneurogenic cells that likely functions as a cell adhesion molecule. Theprotein contains a signal peptide, three immunoglobulin-like domains(Pfam accession PF00047), a transmembrane segment, and a shortcytoplasmic C terminus. IGSF4B is structurally similar to TSLC1, a tumorsuppressor in human non-small cell lung carcinomas, and IGSF4Bexpression is lost or markedly reduced in many glioma cell lines,suggesting that IGSF4B may also function as a tumor suppressor (Fukuharaet al., Oncogene, 20(38):5401-7 (2001); Shingai et al, J Biol Chem,278(37):35421-7 (2003); Fukami et al, Gene 323:11-8 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 17 30 22 69 Expected 17.25 34.5 17.25 69Chi-Sq. = 1.90 Significance = 0.38702 (hom/n) = 0.32 Avg. Litter Size =7Mutation Type: Homologous Recombination (standard)Coding exon 1 was targeted (NCBI accession NM_(—)053199.2).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except brain and eye.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.7.1. Phenotypic Analysis (for Disrupted Gene: DNA35918-1174 (UNQ225)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human immunoglobulinsuperfamily, member 4B (IGSF4B) resulted in numerous immunologicalabnormalities in (−/−) mice. The (−/−) mice also exhibited enhancedmotor coordination. However, circadian rhythm testing showed decreasedambulation for the (−/−) mice. In addition, the mutant (−/−) mice showeda significant increase in mean serum cholesterol levels and mean serumglucose levels. MicroCT observations indicated decreased 5^(th) lumbarbone measurements. Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

The (−/−) mice exhibited decreased mean total white blood cell, absolutelymphocyte, and absolute monocyte counts when compared with their (+/+)littermates and the historical means.

Analyzed wt/het/hom: 7/4/8

Flourescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

The (−/−) mice exhibited an increased mean percentage of CD4 cells and adecreased mean percentage of B cells when compared with their (+/+)littermates and the historical means.

Analyzed wt/het/hom: 7/4/8

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmuno-dominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample. Analyzed wt/het/hom:7/4/8

Results of this challenge: The (−/−) mice exhibited a decreased(virtually absent) mean serum IgG2a response to the ovalbumin challengewhen compared with their (+/+) littermates. Thus, these knockout miceexhibited an decreased ability to elicit an OVA-specific antibodyresponse to the T-cell dependent OVA antigen more than likely due to adefect in Th cells. PRO258 polypeptides or agonists thereof wouldtherefore be expected to stimulate the immune system and would findutility in the cases wherein this effect would be beneficial to theindividual such as in the case of leukemia, and other types of cancer,and in immunocompromised patients, such as AIDS sufferers. Accordingly,inhibitors or antagonists of PRO258 polypeptides would play a role ininhibiting the immune response and would be useful candidates forsuppressing harmful immune responses, e.g. in the case of graftrejection or graft-versus-host diseases.

Overall Summary of Immunological Observations

In summary, the ovalbumin challenge studies, hematology and FACS resultsindicate that the homozygous mutant mice have an impaired immune system,especially in view of the decreased total white blood cell count andabsolute lymphocyte and monocyte counts as well as an inability toelicit an OVA-specific antibody response. These studies all serve as anindicator of a negative phenotype associated with knocking out theDNA35918-1174 gene which encodes PRO258 polypeptides. Thus, PRO258polypeptides and agonists thereof would be important for a healthyimmune system and would be useful in stimulating or inducing the immunesystem's protective functions.

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Circadian Test Description:

Female mice are individually housed at 4 pm on the first day of testingin 48.2 cm×26.5 cm home cages and administered food and water adlibitum. Animals are exposed to a 12-hour light/dark cycle with lightsturning on at 7 am and turning off at 7 pm. The system software recordsthe number of beam interruptions caused by the animal's movements, withbeam breaks automatically divided into ambulations. Activity is recordedin 60, one-hour intervals during the three-day test. Data generated aredisplayed by median activity levels recorded for each hour (circadianrhythm) and median total activity during each light/dark cycle(locomotor activity) over the three-day testing period.

Results:

The (−/−) mice exhibited decreased ambulatory counts during the 1-hourhabituation period of home-cage activity testing when compared withtheir gender-matched (+/+) littermates and the historical mean. Theseresults are indicative of a suppression of circadian rhythm especiallysince there was a marked decrease in the dark period relative to thelittermate controls.

Analyzed wt/het/hom: 6/4/12

Inverted Screen Testing:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Inverted Screen Test Data:

The Inverted Screen is used to measure motor strength/coordination.Untrained mice were placed individually on top of a square (7.5 cm×7.5cm) wire screen which was mounted horizontally on a metal rod. The rodwas then rotated 180 degrees so that the mice were on the bottom of thescreens. The following behavioral responses were recorded over a 1 mintesting session: fell off, did not climb, and climbed up.

Ratio Ratio Genotype Fell Down % Climbed up % +/+ (n = 4) 0/4 0 0/4  0+/− (n = 4) 0/4 0 4/4 100* −/− (n = 8) 1/8 13 7/8  88* *coding indicatesa notable difference.A motor strength deficit is apparent when there is a 50% pointdifference between (−/−) or (+/−) mice and (+/+) mice for the fell downresponse. 0/8 or 1/8 (−/−) or (+/−) mice not climbing indicates impairedmotor coordination. 7/8 or 8/8(−/−) or (+/−) mice climbing up indicatesenhanced motor coordination.

Results:

The Inverted Screen Test is designed to measure basic sensory & motorobservations: Enhanced motor coordination was observed in the (−/−) miceduring inverted screen testing when 7/8 (−/−) mice climbed up thescreen, whereas 0/4 (+/+) mice climbed up.

Analyzed wt/het/hom: 6/4/12

(d) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche). Analyzed wt/het/hom: 4/4/8

Results:

(1) The (−/−) mice exhibited an increased mean serum cholesterol levelwhen compared with their gender-matched (+/+) littermates and thehistorical mean. The mean serum cholesterol values were significantlyabove the normal range. (2) The (−/−) mice also exhibited asignificantly increased mean serum glucose level. During the glucosetolerance test, the (−/−) mice exhibited an increased mean fasting serumglucose level when compared with their gender-matched (+/+) littermatesand the historical mean.

In summary, these knockout mutant mice exhibited a negative phenotypewith regards to lipid metabolism. Thus, mutant mice deficient in thePRO258 gene can serve as a model for treatment of cardiovasculardisease. PRO258 polypeptides, agonists thereof or the encoding gene forPRO258 would be useful in regulating blood lipids and in particular inmaintaining normal cholesterol metabolism. Thus, such agents would beuseful in the treatment of such cardiovascular diseases associated withdyslipidemia as: hypertension, atherosclerosis, heart failure, stroke,various coronary artery diseases, and/or obesity.

In concurrence, knockout mice exhibited the phenotypic pattern of animpaired glucose homeostasis with elevated levels of fasting serumglucose indicative of diabetes or a pre-diabetic condition. Based onthese results, PRO258 (or agonists thereof) or its encoding gene wouldbe useful in the treatment of an impaired glucose metabolism and/ordiabetes.

(e) Bone Metabolism & Body Diagnostics: Bone Metabolism: RadiologyPhenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro-CT: The (−/−) mice exhibited decreased bone measurements in the5^(th) lumbar vertebrae (decreased mean vertebral trabecular bonevolume, number, thickness, and connectivity density) when compared withtheir gender-matched (+/+) littermates and the historical means. Themidshaft femur showed a decreased total area. In addition, the mutant(−/−) mice exhibited a decreased heart rate which was two (2) standarddeviations below the mean.

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO258 polypeptides or agonists thereof play a role in maintaining bonehomeostasis. In addition, PRO258 or its encoding gene would be useful inbone healing or useful for the treatment of arthritis or osteoporosis;whereas antagonists to PRO258 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism such as arthritis,osteoporosis, and osteopenia.

35.8. Generation and Analysis of Mice Comprising DNA39969-1185 (UNQ250)Gene Disruptions

In these knockout experiments, the gene encoding PRO287 polypeptides(designated as DNA39969-1185) (UNQ250) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)029620 or ACCESSION:NM_(—)029620 NID: gi 22095010 ref NM_(—)029620.1 Mus musculusprocollagen C-endopeptidase enhancer 2 (Pcolce2); protein reference:NP_(—)083896 or ACCESSION: NP_(—)083896 NID: gi 22095011 refNP_(—)083896.1 (NM_(—)029620) procollagen C-endopeptidase enhancer 2[Mus musculus]; the human gene sequence reference: NM_(—)013363 orACCESSION: NM_(—)013363 NID: gi 16904386 ref NM_(—)013363.2 Homo sapiensprocollagen C-endopeptidase enhancer 2 (PCOLCE2); the human proteinsequence corresponds to reference: NP_(—)037495 or ACCESSION:NP_(—)037495 NID: gi 7019483 ref NP_(—)037495.1 (NM_(—)013363)procollagen C-endopeptidase enhancer 2 [Homo sapiens].

The mouse gene of interest is Pcolce2 (procollagen C-endopeptidaseenhancer 2), ortholog of human PCOLCE2. Aliases include PCPE2.

PCOLCE2 is a secreted glycoprotein that binds with the C terminus oftype I procollagen, enhancing cleavage of procollagen by C-proteinasessuch as bone morphogenic protein-1. Trabecular meshwork, lungs, heart,brain, liver, skeletal muscle, kidney, pancreas, and placenta expressPCOLCE2 mRNA; however, PCOLCE2 protein is detected mainly in trabecularnetwork. PCOLCE2 is likely to play a role in cartilage formation indifferent tissues during development (Steiglitz et al., J Biol Chem,277(51):49820-30 (2002); Xu et al., Genomics, 66(3):264-73 (2000)).

Genetics Information:

wt het hom Total Observed 25 39 18 82 Expected 20.5 41 20.5 82 Chi-Sq. =1.39 Significance = 0.49901 (hom/n) = 0.22 Avg. Litter Size = 8

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 1 and 2(NCBI accession NM_(—)029620.1).Wild-type expression of the target gene was detected in brain, spinalcord, eye, thymus, spleen, and lung among the 13 adult tissue samplestested by RT-PCR.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-201).

35.8.1. Phenotypic Analysis (for Disrupted Gene: DNA39969-1185 (UNQ250)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human procollagenC-endopeptidase enhancer 2 (PCOLCE2) resulted in growth retardation anddecreased bone measurements in (−/−) mice. Increased uric acid levelsand decreased serum phosphate levels were also observed in the mutant(−/−) mice. Transcript was absent by RT-PCR.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited decreased mean body weight and mean body lengthwhen compared with their gender-matched (+/+) littermates and thehistorical means. Analyzed wt/het/hom: 37/54/28

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean total tissue mass and leanbody mass when compared with their gender-matched (+/+) littermates andthe historical means. These mutant mice also exhibited decreased meanbone mineral content and bone mineral density.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density and decreasedmean femoral mid-shaft cross-sectional area when compared with theirgender-matched (+/+) littermates and the historical means. Analyzedwt/het/hom: 4/4/8

In addition, the (−/−) mice had a significant decrease in body fat.

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. This in conjunction withblood chemistry analysis (see below) wherein increased uric acid levelsand decreased serum phosphate levels were observed. Thus, it appearsthat PRO287 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO287 or its encoding gene would be importantin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO287 would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis, and osteopenia. Inaddition, the (−/−) mice analyzed by DEXA exhibited notably decreasedtotal tissue mass and lean body mass wand decreased body fat whencompared with their (+/+) littermates, suggestive of growth retardationin these mutants. This in conjunction with the observations of decreasedbody weight and length suggest a tissue wasting condition such ascachexia or other growth disorder. Thus, PRO287 polypeptides or agoniststhereof would be useful in the treatment or prevention of growthdisorders such as cachexia and/or other tissue wasting diseases.

(c) Blood Chemistry—Uric Acid and Serum Phosphate Levels

Blood chemistry analysis was performed using the COBAS Integra 400 (mfr:Roche) in its clinical settings for running blood chemistry tests onmice.

Results: Mutant (−/−) mice exhibited increased uric acid levels as wellas decreased serum phosphate levels compared to their control wild-typelittermates.

35.9. Generation and Analysis of Mice Comprising DNA40587-1231 (UNQ289)Gene Disruptions

In these knockout experiments, the gene encoding PRO328 polypeptides(designated as DNA40587-1231) (UNQ289) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)023734 or ACCESSION:NM_(—)023734 NID: 12963802 Mus musculus Mus musculus RIKEN cDNA1200009H11 gene (1200009H11Rik); protein reference: Q9ET66 or ACCESSION:Q9ET66 NID: Mus musculus (Mouse). CYSTEINE-RICH PROTEASE INHIBITOR.MOUSESPTRNRDB; the human gene sequence reference: NM_(—)153370 or Homosapiens protease inhibitor 16 (PI16); the human protein sequencecorresponds to reference: Q8NBK0 or ACCESSION: Q8NBK0 NID: Homo sapiens(Human). Hypothetical protein PLACE1010482.

The mouse gene of interest encodes a hypothetical secreted protein,which is the ortholog of human PI16 (protease inhibitor 16).

PI16 contains a signal peptide, an SCP-like extracellular protein (SCP)domain, and several internal repeats. SCP domains are found inextracellular proteins from many different species. Examples includeinsect venom allergens, mammalian testis-specific proteins, and plantpathogenesis-related proteins (Pfam accession PF00188). PI16 is ahomolog of protease inhibitor 15 (PI15), which also contains an SCPdomain and functions as a secreted serine protease (trypsin) inhibitor(Yamakawa et al., Biochim Biophys Acta, 1395(2):202-8 (1998)). Thefunction of PI16 has not been demonstrated experimentally.

Genetics Information:

wt het hom Total Observed 16 45 22 83 Expected 20.75 41.5 20.75 83Chi-Sq. = 1.46 Significance = 0.48243 (hom/n) = 0.27 Avg. Litter Size =8

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 2 and 3(NCBI accession NM_(—)023734.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-97).

35.9.1. Phenotypic Analysis (for Disrupted Gene: DNA40587-1231 (UNQ289)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human protease inhibitor16 (PI16) resulted in a immunological phenotype in (−/−) mice. Anenhanced glucose tolerance was also observed during the glucosetolerance testing for the (−/−) mice. Changes in lumbar 5 vertebrae werealso noted (decreased measurements compared to littermate controls).Transcript was absent by RT-PCR.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology:

The (−/−) mice exhibited an increased mean red cell distribution widthwhen compared with their (+/+) littermates and the historical mean.

Analyzed wt/het/hom: 9/4/14

Flourescence-Activated Cell-Sorting (FACS) Analysis/Tissue Specific FACS

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS: The (−/−) mice exhibited a decreased meanpercentage of CD21Hi CD23Med cells in the spleen and lymph nodes.Increased mean percentages of CD25+ cells in both spleen and lymph nodeswere also noted in the (−/−) mice.

Analyzed wt/het/hom: 9/4/14

In summary, knocking out DNA40587-1231 (the gene which encodes PRO328polypeptides) resulted in a a decrease in a subset of B cells—marginalzone B cells—that contain a pool of memory cells and participate in fastimmune responses. Thus, antagonists or inhibitors of PRO328 polypeptideswould be expected to demonstrate the same phenotype. PRO328 polypeptideswould be useful in the development or production of marginal zone Bcells useful for participating in fast immune responses.

(c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/hom: 4/4/8

Results:

The (−/−) mice exhibited an enhanced glucose tolerance when comparedwith their gender-matched (+/+) littermates and the historical means.Thus, knockout mice exhibited the opposite phenotypic pattern of animpaired glucose homeostasis, and as such antagonists to PRO328 or itsencoding gene would be useful in the treatment of impaired glucosehomeostasis and diseases associated with abnormal glucose metabolism.

(d) Bone Metabolism & Body Diagnostics: Bone Metabolism: RadiologyPhenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density and decreasedmean femoral mid-shaft cross-sectional area when compared with theirgender-matched (+/+) littermates and the historical means. Analyzedwt/het/hom: 4/4/8

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO328 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO328 or its encoding gene would be importantin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO328 would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis, and osteopenia.

35.10. Generation and Analysis of Mice Comprising DNA40592-1242 (UNQ303)Gene Disruptions

In these knockout experiments, the gene encoding PRO344 polypeptides(designated as DNA40592-1242) (UNQ303) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: BC023068 or Mus musculus C1q andtumor necrosis factor related protein 5; protein reference: Q8R002 orACCESSION: Q8R002NID: Mus musculus (Mouse). Similar to DKFZP586B0621protein (Hypothetical protein); the human gene sequence reference:NM_(—)015645 or Homo sapiens C1q and tumor necrosis factor relatedprotein 5 (C1QTNF5); the human protein sequence corresponds toreference: Q9BXJ0 or ACCESSION: Q9BXJ0 NID: Homo sapiens (Human).Complement C1q tumor necrosis factor-related protein 5 precursor.

The disrupted mouse gene is C1qtnf5 (C1q and tumor necrosis factorrelated protein 5), which is the ortholog of human C1QTNF5. Aliasesinclude CTRP5 and complement-c1q tumor necrosis factor-related protein5.

C1QTNF5 is a hypothetical secreted protein, containing a signal peptide,a collagen triple helix repeat (Pfam accession PF01391), and acomplement component C1q domain (SMART accession SM00110). C1q domainsare globular structures found in many collagens and in the C1 enzymecomplex that activates the serum complement system. The fold of thisdomain is similar to that of tumor necrosis factor. The molecularfunction and biological role of C1QTNF5 is not known.

Genetics Information:

wt het hom Total Observed 19 31 16 66 Expected 16.5 33 16.5 66 Chi-Sq. =0.52 Significance = 0.77292 (hom/n) = 0.24 Avg. Litter Size = 7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred between coding exons 1 and 2 (Accession:NM_(—)145613.2).Wild-type expression of the target gene was detected in brain, spinalcord, eye, lung, kidney, skeletal muscle, and heart among the 13 adulttissue samples tested by RT-PCR. The larger bands are unsplicedgene-specific transcripts.PCR analysis revealed that the transcript was absent in the (−/−) mouseanalyzed (M-131).

35.10.1. Phenotypic Analysis (for Disrupted Gene: DNA40592-1242 (UNQ303)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human C1q and tumornecrosis factor related protein 5 (C1QTNF5) resulted in retinaldegeneration in (−/−) mice. In addition, the (−/−) mice exhibitedabnormal bone measurements with decreased measurements in lumbar 5. Theknockout mice also showed a significant decrease in total body fat andshowed increased uric acid levels. Transcript was absent by RT-PCR.

(b) Cardiovascular Phenotypic Analysis:

In the area of cardiovascular biology, phenotypic testing was performedto identify potential targets for the treatment of cardiovascular,endothelial or angiogenic disorders. One such phenotypic test includedoptic fundus photography and angiography to determine the retinalarteriovenous ratio (A/V ratio) in order to flag various eyeabnormalities. An abnormal A/V ratio signals such systemic diseases ordisorders that may be related to the vascular disease of hypertension(and any disease that causes hypertension, e.g. atherosclerosis),diabetes or other ocular diseases corresponding to ophthalmologicaldisorders. Such eye abnormalities may include but are not limited to thefollowing: retinal abnormality is retinal dysplasia, variousretinopathies, restenosis, retinal artery obstruction or occlusion;retinal degeneration causing secondary atrophy of the retinalvasculature, retinitis pigmentosa, macular dystrophies, Stargardt'sdisease, congenital stationary night blindness, choroideremia, gyrateatrophy, Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Optic fundus photography was performed onconscious animals using a Kowa Genesis small animal fundus cameramodified according to Hawes and coauthors (Hawes et al., 1999 MolecularVision 1999; 5:22). Intra-peritoneal injection of fluorescein permittedthe acquisition of direct light fundus images and fluorescent angiogramsfor each examination. In addition to direct ophthalmological changes,this test can detect retinal changes associated with systemic diseasessuch as diabetes and atherosclerosis or other retinal abnormalities.Pictures were provided of the optic fundus under normal light. Theangiographic pictures allowed examination of the arteries and veins ofthe eye. In addition an artery to vein (A/V) ratio was determined forthe eye.

Ophthalmology analysis was performed on generated F2 wild type,heterozygous, and homozygous mutant progeny using the protocol describedabove. Specifically, the A/V ratio was measured and calculated accordingto the fundus images with Kowa COMIT+ software. This test takes colorphotographs through a dilated pupil: the images help in detecting andclassifying many diseases. The artery to vein ratio (A/V) is the ratioof the artery diameter to the vein diameter (measured before thebifurcation of the vessels). Many diseases will influence the ratio,i.e., diabetes, cardiovascular disorders, papilledema, optic atrophy orother eye abnormalities such as retinal degeneration (known as retinitispigmentosa) or retinal dysplasia, vision problems or blindness. Thus,phenotypic observations which result in an increased artery-to-veinratio in homozygous (−/−) and heterozygous (+/−) mutant progeny comparedto wild-type (+/+) littermates would be indicative of such pathologicalconditions.

Results:

Fundus: Of the 7 non-albino (−/−) mice analyzed, 6 (F-134, F-155, F-158,M-164, M-171, and F-183) exhibited multiple white spots covering theentire retina with attenuated retinal arteries, suggesting retinaldegeneration in the mutants similar to flecked retinal disease inhumans. Analyzed wt/het/hom: 4/4/8

Pathological microscopic observations showed signs of retinaldegeneration in the 4 (−/−) mice analyzed (M-173, M-188, F-197, andF-199). Histological changes were noted wherein the retina showedincreased apoptotic cells in the outer nuclear layer. Gene expressionanalysis could not be performed for this project. In summary, in thisstudy, optic fundus photography showed that (−/−) mice exhibited signsof severe retinal degeneration, namely notably attenuated retinalvessels when compared with their (+/+) littermates. Angiogramsdemonstrated that the mutant (−/−) mice showed attenuated retinalvessels with micro aneurysms. Likewise, microscopic observations showedbilateral retinal degeneration in the mutant (−/−) mice. In summary, byknocking out the gene identified as DNA40592-1242 encoding PRO344polypeptides, homozygous mutant progeny exhibit phenotypes which areassociated with retinal degeneration. Such detected retinal changes aremost commonly associated with cardiovascular systemic diseases ordisorders that may be related to the vascular disease of hypertension(and any disease that causes hypertension, e.g. atherosclerosis),diabetes or other ocular diseases corresponding to ophthalmologicaldisorders such as retinal degeneration. Thus, antagonists of PRO344encoding genes would lead to similar pathological retinal changes,whereas agonists would be useful as therapeutic agents in the treatmentof hypertension, atherosclerosis or other opthamological disordersincluding retinal degeneration and diseases associated with thiscondition (as indicated above).

(c) Bone Metabolism & Body Diagnostics: Bone Metabolism: RadiologyPhenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mutant mice showed a significant decrease in total bodyfat content compared to their littermate controls. In addition, uricacid levels were elevated in the (−/−) mutant mice (as observed in bloodchemistry analysis).

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density and decreasedmean femoral mid-shaft cross-sectional area when compared with theirgender-matched (+/+) littermates and the historical means. Analyzedwt/het/hom: 4/4/8

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO344 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO344 or its encoding gene would be importantin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO344 would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including osteoarthritis, osteoporosis, and osteopenia.In addition, the decrease in total body fat in the (−/−) mice wasindicative of a tissue wasting disease.

35.11. Generation and Analysis of Mice Comprising DNA44804-1248 (UNQ314)Gene Disruptions

In these knockout experiments, the gene encoding PRO357 polypeptides(designated as DNA44804-1248) (UNQ312) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)139307 or Mus musculusSlit-like 2 (Drosophila) (Slit12); protein reference: NP_(—)647468 orSlit-like 2 [Mus musculus]; the human gene sequence reference: BC013767or ACCESSION: BC013767 NID: 15489338 Homo sapiens Homo sapiens, Similarto RIKEN cDNA 2610528G05 gene, clone IMAGE: 3875837; the human proteinsequence corresponds to reference: Q96CX1 or ACCESSION: Q96CX1 NID: Homosapiens (Human). SIMILAR TO RIKEN cDNA 2610528G05 GENE (FRAGMENT).HUMANSPTRNRDB.

The disrupted mouse gene is Slit12 (Slit-like 2 [Drosophila]), theortholog of human hypothetical protein LOC114990. Aliases includehypothetical protein BC013767 and 2610528G05Rik.

Slit-type proteins (e.g., SLIT1) are extracellular proteins that play acritical role in developmental processes, especially nervous andendocrine system formation. Slit-type proteins are thought to assist incellular movement (Piper and Little, Bioessays, 25(1):32-8 (2003)).Thus, by homology, LOC114990 is likely secreted or mostly extracellularand assists in developmental processes.

Genetics Information:

wt het hom Total Observed 15 33 13 61 Expected 15.25 30.5 15.25 61Chi-Sq. = 0.54 Significance = 0.76300 (hom/n) = 0.21 Avg. Litter Size =4

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion disrupted the gene in coding exon 2 (Accession:NM_(—)139307).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and, among the 13 adult tissue samples tested by RT-PCR, inkidney, skeletal muscle, and heart.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (F-59). Disruption of the target gene was confirmed byInverse PCR.

35.11.1. Phenotypic Analysis (for Disrupted Gene: DNA44804-1248 (UNQ314)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalprotein (BC013767) resulted in an increased mean bone mineral content,volumetric bone mineral density and bone mineral density in total body,femur and vertebrae. Several (−/−) mice were smaller than their (+/+)littermates and died early (by 19 days after birth). The (+/−) mice alsoshowed diminished size and died early. Male (−/−) mice exhibitedinfertility. The knockout mice showed increase in serum triglycerides,ketone bodies and glucose levels and had elevated blood pressure. The(−/−) mice also showed increased bone measurements and increased totalbody fat. Transcript was absent by RT-PCR.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Several (−/−) mice were smaller than their (+/+) littermates, and 2 diedby 19 days after birth. The remaining (−/−) mice seemed healthy. Anumber of (+/−) mice were also small, exhibited reduced organ weightsand died early. The 2 male (−/−) mice available for analysis exhibitedincreased mean systolic blood pressure when compared with theirgender-matched (+/+) littermates and the historical mean. Analyzedwt/het/hom: 14/27/10

Fertility: The male (−/−) mouse analyzed (M-106) was infertile.

Pathology: Microscopic observations for the single (−/−) mouse availablefor analysis (M-106) showed a sero purulent exudate from the middle earon one side. Additionally, a cyst was noted on the testis along withdegeneration unilaterally. Analyzed wt/het/hom: 0/2/1

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited increased mean bone mineral content,volumetric bone mineral density, and bone mineral density in total body,femur, and vertebrae when compared with their gender-matched (+/+)littermates and the historical means. The mutant mice also showedincreased total body fat content and a corresponding increase in bloodtriglycerides indicative of dyslipidemia. Analyzed wt/het/hom: 4/8/8

MicroCT: The (−/−) mice increased lumbar 5 vertebrae measurementscompared to their wild-type littermates.

Summary

Several (−/−) mice showed signs of growth retardation since severalmutant mice were much smaller than their (+/+) littermates. In addition,the heterozygous (+/−) mice showed signs of decreased growth and reducedviability being small and not surviving as long as their (+/+)littermates. Furthermore, the (−/−) mice exhibited increased mean bonemineral content, volumetric bone mineral density and total body andfemoral bone mineral density when compared with their gender-matched(+/+) littermates. These results indicate that the knockout mutants notonly showed signs of growth deficiencies and reduced viability but alsoexhibited bone abnormalities associated with such bone diseases asosteopetrosis. Osteopetrosis is a condition characterized by abnormalthickening and hardening of bone and abnormal fragility of the bones. Assuch, PRO357 polypeptides or agonists thereof would be important fornormal growth and normal bone metabolism and would be useful for thetreatment of osteopetrosis or other related bone disorders. In addition,the male (−/−) mice showed signs of infertility and degenerate testes.Thus, PRO357 polypeptides or agonists thereof would be useful in theprevention and/or treatment of such bone disorders and also would play arole in maintaining normal growth and development.

(c) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche).

Results:

The (−/−) mice exhibited increased mean serum triglyceride, ketonebodies and glucose levels when compared with their gender-matched (+/+)littermates and the historical means. In addition, the (−/−) miceexhibited elevated blood pressure.

As summarized above, the (−/−) mice exhibited notably increasedtriglyceride levels when compared with their gender-matched (+/+)littermates and the historical means for the male (+/+) mice. Inaddition, the increased mean serum glucose levels suggesting diabetes.The elevated mean systolic blood pressure along with the blood chemistryis indicative of hypertension or other cardiovascular disease. Thus,mutant mice deficient in the PRO357 gene can serve as a model forcardiovascular disease including diabetes. PRO357 polypeptides or itsencoding gene would be useful in regulating normal blood lipid levelssuch as triglycerides and/or blood sugars. Thus, PRO357 polypeptides oragonists thereof would be useful in the treatment of such cardiovasculardiseases as hypertension, atherosclerosis, heart failure, stroke,various coronary diseases, hypercholesterolemia, hypertriglyceridemia,diabetes and/or obesity.

35.12. Generation and Analysis of Mice Comprising DNA44184-1319 (UNQ330)Gene Disruptions

In these knockout experiments, the gene encoding PRO526 polypeptides(designated as DNA44184-1319) (UNQ330) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)022982 or ACCESSION:NM_(—)022982 NID: gi 12667793 ref NM_(—)022982.1 Mus musculus reticulon4 receptor (Rtn4r); protein reference: Q99PI8 or Reticulon 4 receptorprecursor (Nogo receptor) (NgR) (Nogo-66 receptor); the human genesequence reference: NM_(—)023004 or Homo sapiens reticulon 4 receptor(RTN4R); the human protein sequence corresponds to reference: Q9BZR6 orReticulon 4 receptor precursor (Nogo receptor) (NgR) (Nogo-66 receptor)(UNQ330/PRO526).

The mouse gene of interest is Rtn4r (reticulon 4 receptor), ortholog ofhuman RTN4R. Aliases include NgR, NOGOR, nogo receptor, and Nogo-66receptor.

RTN4R is a glycosylphosphatidylinositol-anchored extracellular proteinthat functions as a receptor for reticulon 4 (RTN4; also known as NOGO),oligodendrocyte myelin glycoprotein (OMG), and myelin-associatedglycoprotein (MAG), which mediate inhibition of axonal growth (Fournieret al., Nature, 409(6818):341-6 (2001); Wang, Koprivica et al., Nature,417(6892):941-4 (2002); Liu et al., Science, 297(5584):1190-3 (2002)).Upon binding with these ligands, RTN4R associates with p75, atransmembrane protein and receptor of the neurotrophin growth factorfamily, and LINGO-1, a nervous system-specific transmembrane protein.This association transduces the signal to the interior of the cell(Wang, Kim et al., Nature, 420(6911):74-8 (2002); Wong et al., NatNeurosci, 5(12):1302-8 (2002); Mi et al., Nat Neurosci, 7(3):221-8(2004)). RTN4R is involved in axonal guidance and nervous systemdevelopment. Moreover, RTN4R and its signaling components are targetsfor inhibitors that enable axonal regeneration following injury to thecentral nervous system (Fisher et al., J Neurosci, 24(7):1646-51 (2004);Song et al., J Neurosci, 24(2):542-6 (2004); Lee et al., Nat Rev DrugDiscov, 2(11):872-8 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 20 36 22 78 Expected 19.5 39 19.5 78 Chi-Sq. =0.56 Significance = 0.75424 (hom/n) = 0.28 Avg. Litter Size = 8Mutation Type: Homologous Recombination (standard)Coding exon 2 was targeted (NCBI accession NM_(—)022982.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.12.1. Phenotypic Analysis (for Disrupted Gene: DNA44184-1319 (UNQ330)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human reticulon 4 receptor(RTN4R) resulted in enhanced glucose tolerance and decreased mean serumcholesterol, triglycerides and glucose levels in (−/−) mice. The male(−/−) mutant mice exhibited an increased lean body mass, increased bonemineral density, and increased bone mineral content in total body,femurs and vertebrae, as well as increased mean body weight. The mutantmice also exhibited a highly elevated TNF-alpha and IL-6 response to theLPS challenge. Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 μl bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACSCalibur instrument.

Results:

The (−/−) mice exhibited a highly elevated mean serum IL-6 and TNF-alpharesponse to LPS challenge when compared with their (+/+) littermates andthe historical mean.

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO526 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited an increased ability to elicit animmunological response (TNF-alpha and IL-6 production) when challengedwith the LPS endotoxin indicating a strong proinflammatory response.IL-6 and TNF-alpha contribute to the later stages of B cell activation.In addition, IL-6 plays a critical role in inducing the acute phaseresponse and systemic inflammation. This suggests that inhibitors orantagonists of PRO526 polypeptides or its encoding gene would stimulatethe immune system and would find utility in the cases wherein thiseffect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immuno-compromised patients,such as AIDS sufferers. Accordingly, PRO526 polypeptides or agoniststhereof thereof would play a role in inhibiting the immune response andwould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The mutant (−/−) mice exhibited a significant increased body weightcompared to their wild-type littermate controls.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (−/−) mice exhibited increased mean lean body mass andincreased mean bone mineral content and bone mineral density in totalbody, femurs, and vertebrae when compared with their gender-matched(+/+) littermates and the historical means.

MicroCT: The (−/−) mice increased trabecular connectivity densitycompared to their wild-type littermates and the historical mean.

Summary

In summary, the male (−/−) mice exhibited increased mean lean body mass,bone mineral content, and total body and femoral bone mineral densitywhen compared with their gender-matched (+/+) littermates. These resultsindicate that the knockout mutant phenotype may be associated with suchbone abnormalities as osteopetrosis. Osteopetrosis is a conditioncharacterized by abnormal thickening and hardening of bone and abnormalfragility of the bones. As such, PRO526 polypeptides or agonists thereofwould be beneficial for the treatment of osteopetrosis or other osteodiseases. On the other hand, inhibitors or antagonists of PRO526polypeptides would be useful in bone healing. Analyzed wt/het/hom: 4/4/8

(d) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche).

Results:

The (−/−) mice exhibited decreased mean serum cholesterol, triglycerideand glucose levels when compared with their gender-matched (+/+)littermates and the historical mean. In summary, these knockout mutantmice exhibited a positive phenotype with regards to lipid and/or glucosemetabolism. Thus, mutant mice deficient in the PRO526 gene can serve asa model for treatment of cardiovascular disease. Antagonists to PRO526or its encoding gene would be useful in regulating blood lipids and inparticular in maintaining normal cholesterol metabolism. Such inhibitorsor antagonists to PRO526 polypeptides would be useful in the treatmentof such cardiovascular diseases associated with dyslipidemia as:hypertension, atherosclerosis, heart failure, stroke, various coronaryartery diseases, obesity and/or diabetes.

(e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/hom: 4/4/8

Results:

The (−/−) mice exhibited an enhanced glucose tolerance when comparedwith their gender-matched (+/+) littermates and the historical means.Thus, knockout mice exhibited the opposite phenotypic pattern of animpaired glucose homeostasis, and as such antagonists to PRO526 or itsencoding gene would be useful in the treatment of impaired glucosehomeostasis and diseases associated with abnormal glucose metabolism.

35.13. Generation and Analysis of Mice Comprising DNA49631-1328 (UNQ389)Gene Disruptions

In these knockout experiments, the gene encoding PRO724 polypeptides(designated as DNA49631-1328) (UNQ389) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)022993 or ACCESSION:NM_(—)022993 NID: 12667805 Mus musculus Mus musculus low-densitylipoprotein receptor-related protein 10 (Lrp10); protein reference:Q9EPE8 or ACCESSION: Q9EPE8 NID: Mus musculus (Mouse). LOW-DENSITYLIPOPROTEIN RECEPTOR-RELATED PROTEIN 9. MOUSESPTRNRDB; the human genesequence reference: NM_(—)014045 or ACCESSION: NM_(—)014045 NID:13027587 Homo sapiens Homo sapiens DKFZP564C1940 protein(DKFZP564C1940); the human protein sequence corresponds to reference:Q86T02. ACCESSION: Q86T02 NID: Homo sapiens (Human). Human full-lengthcDNA clone CSODK002YO06 of HeLa cells of Homo sapiens (Human).

The mouse gene of interest is Lrp10 (low-density lipoproteinreceptor-related protein 10), ortholog of human LRP10. Aliases includeLrp9, MGC8675, and DKFZP564C1940.

LRP10 is a predicted type I plasma membrane protein expressed primarilyin liver that mediates the uptake of apolipoprotein E-enriched beta-VLDLin vitro and, thus, likely functions as a receptor for low-densitylipoproteins in vivo. In addition to liver, LRP10 is also found inkidney and brain, with particularly high levels occurring in vascularwalls. LRP10 may play a role in apoE-containing lipoprotein uptake(Sugiyama et al., Biochemistry, 39(51):15817-25 (2000)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 17 29 17 63 Expected 15.75 31.5 15.75 63Chi-Sq. = 0.40 Significance = 0.82003 (hom/n) = 0.27 Avg. Litter Size =6Mutation Type: Homologous Recombination (standard)Coding exons 3 through 7 were targeted (NCBI accession NM_(—)022993.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.13.1. Phenotypic Analysis (for Disrupted Gene: DNA49631-1328 (UNQ389)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human low-densitylipoprotein receptor-related protein 10 (LRP10) resulted in adramatically decreased skin fibroblast proliferation rate in (−/−) mice.In addition, (−/−) mice exhibited increased bone measurements. Animpaired glucose tolerance was also noted for the mutant mice. Also, themutant (−/−) mice exhibited immunological abnormalities marked bydecreased levels of eosinophils and monocytes but also increased levelsof CD8+ cells. Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

(1) Flourescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

(2) Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

The (−/−) mice exhibited decreased eosinophils and monocytes whencompared with their (+/+) littermates and the historical means.

FACS analysis also demonstrated that (−/−) mice exhibit an increasedmean percentage of CD8+ cells when compared with their (+/+) littermatesand the historical mean.

Analyzed wt/het/hom: 7/5/8

In summary, the hematology and FACS results indicate that the homozygousmutant mice exhibited decreased eosinophil and monocyte counts comparedto their littermate controls indicating low levels of precursors ofmacrophages. However, the (−/−) mutant mice also showed an increasedpercentage of CD8+ cells. CD8+ molecules are the co-receptor moleculeswhich cooperate with the T-cell receptor in antigen recognition and inparticular specifically bind only to the invariant parts of the MHCclass I molecule. During antigen recognition, the CD8+ moleculesassociate on the T-cell surface with components of the T-cell receptorto form the cytotoxic CD8+ T-cell. Thus, inhibitors or antagonists ofPRO724 polypeptides would be important in the T-cell mediated responseinvolving the MHC class I pathway and would be beneficial in thoseinstances wherein cytotoxic T cells are required in host defense againstcytosolic pathogens. In contrast, PRO724 polypeptides or agoniststhereof, would be expected to mimic a negative phenotype resulting in adeficiency in the mean percentage of CD8+ cells and therefore an MHCclass I deficiency would result. One such disease model occurs whenthere is an almost complete absence of cell-surface MHC class Imolecules. Patients with this condition have normal levels of mRNAencoding MHC class I molecules and normal levels of production of MHCclass I proteins. However, these individuals are immunodeficient,specifically owing to the lack of CD8+ T cells. This results in a severeimmunodeficiency disease wherein the response to nearly all pathogens iscritically suppressed.

(c) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited increased mean bone mineral density whencompared with their gender-matched (+/+) littermates and the historicalmeans.

Micro-CT: The (−/−) mice exhibited increased mean vertebral trabecularbone volume, number, and connectivity density when compared with theirgender-matched (+/+) littermates and the historical means.

Analyzed wt/het/hom: 4/4/9

In summary, the (−/−) mice exhibited increased mean bone mineralcontent, and total body and femoral bone mineral density when comparedwith their gender-matched (+/+) littermates. These results indicate thatthe knockout mutant phenotype is associated with bone abnormalities suchas osteopetrosis. Osteopetrosis is a condition characterized by abnormalthickening and hardening of bone and abnormal fragility of the bones. Assuch, PRO724 polypeptides or agonists thereof would be beneficial forthe treatment of osteopetrosis or other osteo diseases. On the otherhand, inhibitors or antagonists of PRO724 polypeptides would be usefulin bone healing.

(d) Oncology Phenotypic Analysis

In the area of oncology, targets were identified herein for thetreatment of solid tumors, lymphomas and leukemia.

Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

The (−/−) mice exhibited a dramatically decreased mean skin fibroblastproliferation rate when compared with their gender-matched (+/+)littermates and the historical mean. Thus, homozygous mutant micedemonstrated a hypo-proliferative phenotype. As suggested by theseobservations, antagonists of a PRO724 polypeptide or its encoding genewould be useful in decreasing abnormal cell proliferation such as tumorcell growth.

(e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygote mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection.

Results:

The (−/−) mice exhibited an increased mean serum glucose level whencompared with their gender-matched (+/+) littermates and the historicalmean. In addition, the (−/−) mice exhibited impaired glucose tolerancewhen compared with their gender-matched (+/+) littermates and thehistorical means.

Analyzed wt/het/hom: 4/4/8

These studies indicated that (−/−) mice exhibit a decreased glucosetolerance in the presence of normal fasting glucose at all 3 intervalstested when compared with their gender-matched (+/+) littermates and thehistorical means. Thus, knockout mutant mice exhibited the phenotypicpattern of an impaired glucose homeostasis, and therefor PRO724polypeptides (or agonists thereof) or its encoding gene would be usefulin the treatment of impaired glucose homeostasis and/or variouscardiovascular diseases, including diabetes.

35.14. Generation and Analysis of Mice Comprising DNA48331-1329 (UNQ395)Gene Disruptions

In these knockout experiments, the gene encoding PRO731 polypeptides(designated as DNA48331-1329) (UNQ395) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)017378 or ACCESSION:NM_(—)017378 NID: 8393915 Mus musculus Mus musculus protocadherin 12(Pcdh12); protein reference: O55134 or ACCESSION: O55134 NID: Musmusculus (Mouse). VASCULAR CADHERIN-2. MOUSESPTRNRDB; the human genesequence reference: NM_(—)016580 or ACCESSION: NM_(—)016580 NID:14589925 Homo sapiens Homo sapiens protocadherin 12 (PCDH12); the humanprotein sequence corresponds to reference: Q9NPG4 or ACCESSION: Q9NPG4NID: Homo sapiens (Human). Protocadherin 12 precursor (Vascularcadherin-2) (Vascular endothelial cadherin-2) (VE-cadherin-2)(VE-cad-2). HUMANSPTRNRDB.

The mouse gene of interest is Pcdh12 (protocadherin 12), ortholog ofhuman PCDH12. Aliases include Pcdh14, VE-cad-2, VE-cadherin-2, VECAD2,protocadherin 14, and vascular endothelial cadherin-2.

PCDH12 is a type I membrane protein expressed in vascular endotheliumthat likely functions as a cadherin family cell adhesion molecule. Theprotein consists of an extracellular domain containing six cadherinrepeats, a transmembrane segment, and a cytoplasmic C terminus. Unlikevascular endothelial cadherin-1, PCDH12 neither interacts with cateninsnor affects cell migration or growth. PCDH12 promotes homotypiccalcium-dependent adhesion and aggregation clusters at intercellularjunctions (Telo et al., J Biol Chem 273(28):17565-72 (1998); Wu andManiatis, Proc Natl Acad Sci USA, 97(7):3124-9 (2000)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 20 40 19 79 Expected 19.75 39.5 19.75 79Chi-Sq. = 0.04 Significance = 0.98119 (hom/n) = 0.24 Avg. Litter Size =8Mutation Type: Homologous Recombination (standard)Coding exon 1 was targeted (NCBI accession NM_(—)017378.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except stomach, small intestine, andcolon.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.14.1. Phenotypic Analysis (for Disrupted Gene: DNA48331-1329 (UNQ395)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human protocadherin 12(PCDH12) resulted in larger (−/−) mice, exhibiting increased bodyweight, total tissue mass, and lean body mass as well increased bonerelated measurements. The (−/−) mice exhibited increased organ weight,total body fat and total fat mass. In addition, the (−/−) mice exhibiteda decreased percentage of natural killer cells and blood eosinophils.Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

Flourescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

FACS: The (−/−) mice exhibited a decreased mean percentage of naturalkiller cells and blood eosinophils when compared with their (+/+)littermates and the historical mean. Analyzed wt/het/hom: 7/4/8

In summary, the FACS results indicate that the homozygous mutant micehave an impaired immune system, especially in view of the decreased meanpercentage of natural killer cells which is an indicator of a negativephenotype associated with knocking out the DNA48331-1329 gene whichencodes PRO731 polypeptides. Natural killer cells are the first line ofdefense to viral infection since these cells have been implicated inviral immunity and in defense against tumors. Natural killer cells or NKcells act as effectors in antibody-dependent cell-mediated cytotoxicityand have been identified by their ability to kill certain lymphoid tumorcell lines in vitro without the need for prior immunization oractivation. However, their known function in host defense is in theearly phases of infection with several intracellular pathogens,particularly herpes viruses. Thus, PRO731 polypeptides and agoniststhereof would be important for a healthy immune system and would beuseful in stimulating the immune system particularly during viralinfections.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited increased mean body weight when compared withtheir gender-matched (+/+) littermates and the historical means. Organweights were also significantly increased. Analyzed wt/het/hom: 28/58/33

Pathology: Microscopic observations revealed apoptosis of the olfactoryneuro epithelial cells in 3/6 (−/−) and 1/2 (+/+) mice.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immuno histochemical analysis. Analyzed wt/het/hom: 4/2/13

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited increased mean total tissue mass, leanbody mass, total body bone mineral density, and volumetric bone mineraldensity when compared with their gender-matched (+/+) littermates andthe historical means. In addition, the (−/−) mice exhibited increasedmean percent total body fat and total fat mass.

Micro-CT: The (−/−) mice exhibited increased mean vertebral trabecularbone volume, number and connectivity density and increased mean femoralmid-shaft cortical thickness and cross-sectional area when compared withtheir gender-matched (+/+) littermates and the historical means.

Analyzed wt/het/hom: 4/4/8

In summary, the (−/−) mice exhibited increased body weight, increasedbody fat, increased mean lean body mass, increased bone mineral content,and increased total body and femoral bone mineral density when comparedwith their gender-matched (+/+) littermates. The observations of anincreased body weight, body fat and mean lean body mass in the (−/−)mutant mice suggests an obesity phenotype. These data suggest that theDNA48331-1329 gene encoding PRO731 polypeptides serves to negativelyregulate proliferation and growth (cell/organ size) and would beimportant for cellular survival. In addition, the abnormal bonemeasurements indicate that the knockout mutant phenotype is associatedwith bone abnormalities such as osteopetrosis. Osteopetrosis is acondition characterized by abnormal thickening and hardening of bone andabnormal fragility of the bones. As such, PRO731 polypeptides oragonists thereof would be beneficial for the treatment of osteopetrosisor other osteo diseases. On the other hand, inhibitors or antagonists ofPRO731 polypeptides would be useful in bone healing.

38.15. Generation and Analysis of Mice Comprising DNA48334-1435 (UNQ396)Gene Disruptions

In these knockout experiments, the gene encoding PRO732 polypeptides(designated as DNA48334-1435) (UNQ396) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019760 or ACCESSION:NM_(—)019760 NID: gi 9790268 ref NM_(—)019760.1 Mus musculus tumordifferentially expressed 1, like (Tde1l); protein reference: Q9QZI8 orACCESSION: Q9QZI8 NID: Mus musculus (Mouse). Tumor differentiallyexpressed 1 protein like (Membrane protein TMS-2). MOUSESPTRNRDB; thehuman gene sequence reference: NM_(—)020755 or ACCESSION: NM_(—)020755NID: gi 24308212 ref NM_(—)020755.1 Homo sapiens likely ortholog ofmouse tumor differentially expressed 1, like (TDE1L); the human proteinsequence corresponds to reference: Q9NRX5 or ACCESSION: Q9NRX5 NID: Homosapiens (Human). Tumor differentially expressed 1 protein like.HUMANSPTRNRDB.

The mouse gene of interest is Tde2 (tumor differentially expressed 2),ortholog of human TDE2. Aliases include Tms2, TMS-2, Tde1l, AIGP2,membrane protein TMS-2, and tumor differentially expressed 1-like.

TDE2 is a putative integral plasma membrane protein expressed in neuronsof the central nervous system (Grossman et al., J Exp Biol 203 Pt3:447-57 (2000)) and in several other tissues, such as bladder, kidney,and muscle (Player et al, Int J Cancer, 107(2):238-43 (2003)). Theprotein consists of several transmembrane segments contained within a“TMS membrane protein/tumor differentially expressed protein (TDE)”domain. This domain is found in several other proteins, constituting afamily that is differentially expressed in various tumor and cell lines(Pfam accession PF03348). For example, TDE2 expression tends to behigher in non small cell lung cancers than in adjacent normal tissue. Incontrast, TDE2 expression in lung tumors tends to be lower than that inadjacent nonmalignant bronchiole epithelium (Player et al., Int JCancer, 107(2):238-43 (2003)). The function of this protein is notknown.

Genetics Information:

wt het hom Total Observed 13 32 9 54 Expected 13.5 27 13.5 54 Chi-Sq. =2.44 Significance = 0.29457 (hom/n) = 0.17 Avg. Litter Size = 6

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 1 and 2(NCBI accession NM_(—)019760.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except bone.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-133). Disruption of the target gene was confirmed byInverse PCR.

35.15.1. Phenotypic Analysis (for Disrupted Gene: DNA48334-1435 (UNQ396)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human tumor differentiallyexpressed 2 (TDE2) resulted in growth retardation as well as boneabnormalities (decreased bone measurements) in (−/−) mice. The (−/−)mice also exhibited an increased mean serum glucose level and impairedglucose tolerance. Hydrocephalus was noted in several of the (−/−)mutant mice. In addition, open field testing revealed increased activityduring open field testing in the mutant (−/−) mice. Transcript wasabsent by RT-PCR.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Both the male and female (−/−) mice exhibited decreased mean body weightand mean body length when compared with their gender-matched (+/+)littermates and the historical means. The difference was more notable inthe males. Analyzed wt/het/hom: 20/45/10

Pathology/Microscopic Observations: Hydrocephalus was noted in several(−/−) mice available for analysis.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis. Analyzed wt/het/hom: 2/2/6

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean total tissue mass and leanbody mass when compared with their gender-matched (+/+) littermates andthe historical means. These mutant animals also exhibited decreased meanbone mineral content and bone mineral density-related measurements.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density and decreasedmean femoral mid-shaft cortical thickness when compared with theirgender-matched (+/+) littermates and the historical means.

Analyzed wt/het/hom: 4/4/8

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO732 or agonists thereof would play a role in maintaining bonehomeostasis. In addition, PRO732 or its encoding gene would be usefulfor maintaining bone homeostasis and for bone healing or for thetreatment of arthritis or osteoporosis; whereas antagonists to PRO732 orits encoding gene would lead to abnormal or pathological bone disordersincluding inflammatory diseases associated with abnormal bone metabolismincluding arthritis, osteoporosis, and osteopenia.

The (−/−) mice analyzed by DEXA exhibited notably decreased total tissuemass and lean body mass when compared with their (+/+) littermates,suggestive of growth retardation in these mutants. This in conjunctionwith the observations of abnormal bone measurements suggest a tissuewasting condition or other growth related disorders such as cachexia.Thus, PRO732 polypeptides or agonists thereof would be useful in thetreatment of bone disorders but also would be useful for the preventionof growth related disorders such as cachexia and/or other tissue wastingdiseases.

(c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection.

Results:

Blood Chemistry: The (−/−) mice exhibited an increased mean serumglucose level when compared with their gender-matched (+/+) littermatesand the historical mean. In the glucose tolerance test: 4 (−/−) miceanalyzed, 2 (M-157 and M-167), exhibited increased fasting serum glucoselevels when compared with their gender-matched (+/+) littermates and thehistorical mean. Analyzed wt/het/hom: 4/5/8

These studies indicated that (−/−) mice exhibit a decreased glucosetolerance in the presence of normal fasting glucose at all 3 intervalstested when compared with their gender-matched (+/+) littermates and thehistorical means. Thus, knockout mice exhibited the phenotypic patternof an impaired glucose homeostasis, and therefor PRO732 polypeptides (oragonists thereof) or its encoding gene would be useful in the treatmentof impaired glucose homeostasis.

(d) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning First, the field (40×40 cm) wasselected to be relatively large for a mouse, thus designed to pick upchanges in locomotor activity associated with exploration. In addition,there were 4 holes in the floor to allow for nose-poking, an activityspecifically related to exploration. Several factors were also designedto heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value. Analyzed wt/het/hom: 5/4/8

Results:

The (−/−) mice exhibited a decreased median sum time-in-center duringopen field testing when compared with their gender-matched (+/+)littermates and the historical mean, suggesting an increasedanxiety-like response in the mutants. As noted above, a notabledifference was observed during open field activity testing. The (−/−)mice exhibited a decreased median sum time in the center area whencompared with their gender-matched (+/+) littermates. This type ofbehavior is consistent with an increased anxiety like response. Thus,the knockout mice demonstrated a phenotype consistent with anxietyrelated disorders which are associated with mild to moderate anxiety,anxiety due to a general medical condition, and/or bipolar disorders;hyperactivity; sensory disorders; obsessive-compulsive disorders,schizophrenia or a paranoid personality. Thus, PRO732 polypeptides oragonists thereof would be useful in the treatment of such neurologicaldisorders or the amelioration of the symptoms associated with anxietydisorders.

35.16. Generation and Analysis of Mice Comprising DNA58846-1409 (UN0487)Gene Disruptions

In these knockout experiments, the gene encoding PRO1003 polypeptides(designated as DNA58846-1409) (UNQ487) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)032541 or ACCESSION:NM_(—)032541 NID: gi 14211541 ref NM_(—)032541.1 Mus musculus hepcidinantimicrobial peptide (Hamp); protein reference: Q9EQ21 or ACCESSION:Q9EQ21 NID: Mus musculus (Mouse). PROHEPCIDIN (HEPCIDIN ANTIMICROBIALPEPTIDE). MOUSESPTRNRDB; the human gene sequence reference: NM_(—)021175or ACCESSION: NM_(—)021175 NID: gi 10863972 ref NM_(—)021175.1 Homosapiens hepcidin antimicrobial peptide (HAMP); the human proteinsequence corresponds to reference: P81172 or ACCESSION: P81172 NID: Homosapiens (Human). ANTIMICROBIAL PEPTIDE HEPCIDIN PRECURSOR(LIVER-EXPRESSED ANTIMICROBIAL PEPTIDE) (LEAP-1) (PUTATIVE LIVER TUMORREGRESSOR) (PLTR) [CONTAINS: HEPCIDIN 25 (HEPC25); HEPCIDIN 20(HEPC20)]. HUMANSPTRNRDB.

The disrupted mouse gene is hepcidin antimicrobial peptide (Hamp),ortholog of human HAMP. Aliases include HEPC1, HEPC, HFE2, LEAP1,LEAP-1, and liver-expressed antimicrobial peptide.

HAMP is a protein secreted mainly from liver that functions as an ironregulatory hormone and mediator of innate immunity The 84-amino acidprotein contains a signal peptide, a propeptide, and a 25-amino acidhepcidin core at the C-terminus. The positively charged hydrophilicresidues and the hydrophobic residues of the mature 25 amino acidhepcidin core peptide are spatially separated, enabling the peptide todisrupt microbial membranes (Ganz Tomas., Blood 102(3):783-8 (2003)).

HAMP is involved in iron homeostasis. Increases in iron load increaseHAMP expression, resulting in decreased dietary iron absorption,transplacental iron transport, and iron mobilization from splenic andhepatic macrophages. Mutations in the HAMP gene can cause juvenilehereditary hemochromatosis, leading to iron overload, cirrhosis,cardiomyopathy, arthritis, and endocrine failure. HAMP has strongantimicrobial activity against bacteria and some fungi. However,individuals lacking active HAMP still have the ability to preventinfection, suggesting that the role of HAMP as an antimicrobial is notcritical (Roetto et al., Nat Genet, 33(1):21-2 (2003)). Inflammationupregulates HAMP, possibly resulting in iron disorders and anemia inresponse to inflammatory diseases such as infectious disease,osteoarticular diseases, and malignancies (Ganz Tomas, Blood,102(3):783-8 (2003)).

Nicolas and colleagues, Proc Natl Acad Sci USA, 98(15):8780-5 (2001)showed that HAMP-deficient mice (caused by knockout of a transcriptionfactor gene that regulates HAMP expression) develop multivisceral ironoverload. In a mouse model of hemochromatosis, Nicolas and colleagues,Nat Genet, 34(1):97-101 (2003) showed that iron accumulation normallyoccurring in these mice was inhibited by overexpression of HAMP. Inwild-type mice, Nicolas and colleagues, Proc Natl Acad Sci USA,99(7):4596-601 (2002) showed that HAMP overexpression produced mice withpale skin that died a few hours after birth. These animals had decreasedbody iron levels and presented severe microcytic hypochromic anemia.

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 20 33 24 77 Expected 19.25 38.5 19.25 77Chi-Sq. = 1.99 Significance = 0.37028 (hom/n) = 0.31 Avg. Litter Size =8Mutation Type: Homologous Recombination (standard)Coding exons 1 and 2 were targeted (NCBI accession NM_(—)032541.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and, in all 13 adult tissues samples tested by RT-PCR, exceptliver, heart and adipose.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.16.1. Phenotypic Analysis (for Disrupted Gene: DNA58846-1409 (UNQ487)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human hepcidinantimicrobial peptide (HAMP) resulted in enhanced glucose tolerance in(−/−) mice. The knockout mice (−/−) also showed elevated levels of serumuric acid. Gene disruption was confirmed by Southern blot.

(b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/horn: 4/4/9

Results:

The (−/−) mice exhibited enhanced glucose tolerance and a significantdecrease in mean serum glucose when compared with their gender-matched(+/+) littermates and the historical means. Thus, knockout miceexhibited the opposite phenotypic pattern of an impaired glucosehomeostasis, and as such antagonists to PRO1003 or its encoding genewould be useful in the treatment of impaired glucose homeostasis and/orany associated metabolic disorder. In addition, the knockout mutant mice(−/−) exhibited elevated serum uric acid levels (greater than 2 SD abovethe historical mean). However, there were no other indices of renalcompromise.

35.17. Generation and Analysis of Mice Comprising DNA59616-1465 (UNQ547)Gene Disruptions

In these knockout experiments, the gene encoding PRO1104 polypeptides(designated as DNA59616-1465) (UNQ547) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)131066 or Mus musculussimilar to hypothetical protein FLJ20519 (LOC229588); protein reference:XP_(—)131066 or similar to hypothetical protein FLJ20519 [Mus musculus];the human gene sequence reference: NM_(—)017860 or Homo sapienshypothetical protein FLJ20519 (FLJ20519); the human protein sequencecorresponds to reference: NP_(—)060330 or hypothetical protein FLJ20519[Homo sapiens].

The disrupted mouse gene is a hypothetical protein (interim name,LOC229588), which is orthologous to human hypothetical protein FLJ20519.

A signal peptide-like region is predicted at the N-terminus throughbioinformatic analysis. Overall, the protein is predicted to be secretedor to reside in the plasma membrane.

Genetics Information:

wt het hom Total Observed 8 19 20 47 Expected 11.75 23.5 11.75 47Chi-Sq. = 7.85 Significance = 0.01973 (hom/n) = 0.43 Avg. Litter Size =7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the first coding exon (Accession:XM_(—)131066).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and, among the 13 adult tissue samples tested by RT-PCR, inspinal cord, eye, thymus, and lung.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-104). Disruption of the target gene was confirmed byInverse PCR.

35.17.1. Phenotypic Analysis (for Disrupted Gene: DNA59616-1465 (UNQ547)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalprotein (FLJ2519) resulted in an increased body weight and lengthmeasurements as well as increased body mass and femoral bonemeasurements. The ovalbumin (OVA) challenge resulted in reduced anti-OVAtiters in the homozygous knockout mice. In addition, the (−/−) miceshowed an enhanced glucose tolerance compared with their wild-typelittermates. Transcript was absent by RT-PCR.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmuno-dominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample. Analyzed wt/het/hom:7/4/8

Results of this challenge: The (−/−) mice exhibited a decreased meanserum IgG2a response to the ovalbumin challenge when compared with their(+/+) littermates. Thus, these knockout mice exhibited an decreasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen more than likely due to a defect in Th cells.PRO1104 polypeptides or agonists thereof would therefore be expected tostimulate the immune system and would find utility in the cases whereinthis effect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immunocompromised patients,such as AIDS sufferers. Accordingly, inhibitors or antagonists ofPRO1104 polypeptides would play a role in inhibiting the immune responseand would be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited increased mean body weight and increased meanbody length (by at least one (1) standard deviation heavier thancontrols before 8 weeks of age) when compared with their gender-matched(+/+) littermates and the historical mean. Analyzed wt/het/hom: 15/22/16

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited increased mean total tissue mass and leanbody mass when compared with their gender-matched (+/+) littermates andthe historical means.

Micro-CT: The (−/−) mice exhibited increased mean femoral mid-shaftcross-sectional area when compared with their gender-matched (+/+)littermates and the historical means.

Analyzed wt/het/hom: 4/4/8

In summary, the (−/−) mice exhibited increased body weight and length,increased mean total tissue mass and lean body mass and increased bonecross-sectional measurements when compared with their gender-matched(+/+) littermates. These observations suggests an obesity and/or growthdisorder type phenotype. In addition, the mutant (−/−) mice exhibited anabnormal bone development. Thus, PRO1104 polypeptides or agoniststhereof, would be useful for normal growth and bone development andwould play a role in the treatment of related growth or metabolicdisorders associated with obesity and/or bone disorders.

(d) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/horn: 4/4/9

Results:

The (−/−) mice exhibited enhanced glucose tolerance and a significantdecrease in mean serum glucose when compared with their gender-matched(+/+) littermates and the historical means. Thus, knockout miceexhibited the opposite phenotypic pattern of an impaired glucosehomeostasis, and as such antagonists to PRO1104 or its encoding genewould be useful in the treatment of impaired glucose homeostasis and/orany associated metabolic disorder.

35.18. Generation and Analysis of Mice Comprising DNA44694-1500 (UNQ581)Gene Disruptions

In these knockout experiments, the gene encoding PRO1151 polypeptides(designated as DNA44694-1500) (UNQ581) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)028331 or Mus musculus C1qand tumor necrosis factor related protein 6 (C1qtnf6); proteinreference: Q8BKR0 or ACCESSION: Q8BKR0 NID: Mus musculus (Mouse). Weaklysimilar to complement-C1Q tumor necrosis factor-related protein; thehuman gene sequence reference: NM_(—)031910 or Homo sapiens C1q andtumor necrosis factor related protein 6 (C1QTNF6), transcript variant 1;the human protein sequence corresponds to reference: NP_(—)114116 or C1qand tumor necrosis factor related protein 6; complement-c1q tumornecrosis factor-related protein 6 [Homo sapiens]gi|32967300|ref|NP_(—)872292.1| C1q and tumor necrosis factor relatedprotein 6; complement-c1q tumor necrosis factor-related protein 6 [Homosapiens] gi|13274531|gb|AAK17966.1| complement-c1q tumor necrosisfactor-related protein [Homo sapiens].

The mouse gene of interest is C1qtnf6 (C1q and tumor necrosis factorrelated protein 6), ortholog of human C1QTNF6. Aliases include CTRP6,ZACRP6, and complement-c1q tumor necrosis factor-related protein 6.

C1QTNF6 is a putative secreted protein, consisting of a signal peptide,a collagen triple helix repeat, and a “complement component C1q” domain.Collagen triple helix repeats are most often found in collagens, whichare generally extracellular structural proteins (Pfam accessionPF01391). C1q domains are found in many collagens and in the C1q subunitof C1 enzyme complex, which activates the serum complement system. C1qand tumor necrosis factor fold similarly (SMART accession SM00110). Thefunction of this protein is not known.

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 18 25 13 56 Expected 14 28 14 56 Chi-Sq. =1.54 Significance = 0.46401 (hom/n) = 0.23 Avg. Litter Size = 8Mutation Type: Homologous Recombination (standard)Coding exon 2 was targeted (NCBI accession NM_(—)028331.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptskeletal muscle.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.18.1. Phenotypic Analysis (for Disrupted Gene: DNA44694-1500 (UNQ581)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human C1q and tumornecrosis factor related protein 6 (C1QTNF6) resulted in decreased bonemineral density measurements. Gene disruption was confirmed by Southernblot.

(b) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean bone mineral density intotal body and femur when compared with their gender-matched (+/+)littermates and the historical means.

Micro-CT: The (−/−) mice exhibited decreased mean femoral midshaftcortical thickness when compared with their gender-matched (+/+)littermates and the historical means.

Analyzed wt/het/hom: 5/4/8

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO1151 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO1151 or its encoding gene would be usefulin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO1151 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis, and osteopenia.

35.19. Generation and Analysis of Mice Comprising DNA64883-1526 (UNQ628)Gene Disruptions

In these knockout experiments, the gene encoding PRO1244 polypeptides(designated as DNA64883-1526) (UNQ628) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)025952 or Mus musculus RIKENcDNA 2610529C04 gene (2610529C04Rik); protein reference: NP_(—)080228 orimplantation-associated protein [Mus musculus]; the human gene sequencereference: NM_(—)032121 or Homo sapiens implantation-associated protein(DKFZp564K142); the human protein sequence corresponds to reference:NP_(—)115497 or implantation-associated protein [Homo sapiens].

The disrupted mouse gene encodes a hypothetical protein, which is theortholog of human FLJ14726. Aliases include 2610529C04Rik, IAG2,2410001C15Rik, PRO0756, DKFZp564K142, implantation-associated protein,and implantation-associated uterine protein.

FLJ14726 contains an OST3/OST6 motif (Pfam PF04756) suggesting that itmight be an oligosaccharide transferase and might be located in theendoplasmic reticulum. Proteins with similar function have beendescribed and studied in yeast (Knauer and Lehle, J Biol Chem,274(24):17249-56 (1999).

This project is X-linked, hemizygotes have no notable phenotype.

Summary of X-linked Gene Distribution by Sex and Genotype

(Only the agouti pups from the male chimeras are included.)

Summary of X-linked Gene Distributions for Sex by Genotype Agouti F1 F1aProgeny (M chimera × wt) Progeny (F het × wt) Sex wt het Sex wt het hemiM 24 0 M 32 n/a 23 F 16 11 F 18 18 n/a

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation.

wt het hom Total Observed 22 18 30 70 Expected 17.5 35 17.5 70 Chi-Sq. =18.34 Significance = 0.00010 (hom/n) = 0.43 Avg. Litter Size = 7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred between coding exons 1 and 2 (Accession:NM_(—)025952).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except skeletal muscle, bone, andadipose.RT-PCR analysis revealed that the transcript was absent in the (0/−)mouse analyzed (M-97).

35.19.1. Phenotypic Analysis (for Disrupted Gene: DNA64883-1526 (UNQ628)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of humanimplantation-associated protein (DKFZp564K142) protein resulted in anincreased mean serum glucose level. Transcript was absent by RT-PCR.

(b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/horn: 4/4/12

Results:

Blood Chemistry: The (−/−) mice exhibited an increased mean serumglucose level when compared with their gender-matched (+/+) littermatesand the historical mean.

Thus, knockout mice exhibited the phenotypic pattern of an impairedglucose homeostasis, and as such PRO1244 polypeptides or agoniststhereof, would be useful in the treatment of impaired glucosehomeostasis and/or any associated metabolic disease.

35.20. Generation and Analysis of Mice Comprising DNA66511-1563 (UNQ666)Gene Disruptions

In these knockout experiments, the gene encoding PRO1298 polypeptides(designated as DNA66511-1563) (UNQ666) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019998 or ACCESSION:NM_(—)019998 NID: 9910439 Mus musculus Mus musculus RIKEN cDNA1300013N08 gene (1300013N08Rik); protein reference: Q9JJA8 or Q9JJA8Q9JJA8 BRAIN cDNA, CLONE MNCB-5081; the human gene sequence reference:NM_(—)033087 or ACCESSION: NM_(—)033087 NID: 14861835 Homo sapiens Homosapiens hypothetical protein FLJ14511 (FLJ14511); the human proteinsequence corresponds to reference: Q9H553 or Q9H553 Q9H553 BA13B9.1NOVEL PROTEIN SIMILAR TO A GLYCOS.

The mouse gene of interest is Alg2 (asparagine-linked glycosylation 2homolog [yeast, alpha-1,3-mannosyltransferase]), ortholog of human ALG2.Aliases include ALPG2, CDGIi, MNCb-5081, hALPG2, FLJ14511, homolog ofyeast ALG2, and GDP-Man:Man(1)GlcNAc(2)-PP-dolichol mannosyltransferase.

ALG2 is an alpha 1,3-mannosyltransferase located in the lumen of theendoplasmic reticulum that catalyzes the formation ofManalpha1,3-ManGlcNAc2-PP dolichol from Man1GlcNAc2-PP-dolichol andGDP-mannose. ALG2 appears to play an important role in glycoproteinbiosynthesis. Loss-of-function mutations in ALG2 cause congenitaldisorder of glycosylation type Ii. Individuals with this mutation arenormal at birth but develop a multisystemic disorder in the first yearof life. The disorder includes mental retardation, seizures, coloboma ofthe iris, hypomyelination, hepatomegaly, and coagulation abnormalities(Thiel et al., J Biol Chem, 278(25):22498-505 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

wt het hom Total Observed 10 37 0 47 Expected 11.75 23.5 11.75 47Chi-Sq. = 19.77 Significance = 0.00005 (hom/n) = 0.00 Avg. Litter Size =5Mutation Type: Homologous Recombination (standard)Coding exon 2 was targeted (NCBI accession NM_(—)019998.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptlung, bone, and adipose.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.20.1. Phenotypic Analysis (for Disrupted Gene: DNA66511-1563 (UNQ666)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human asparagine-linkedglycosylation 2 homolog (ALG2) resulted in lethality of (−/−) mutants.The (+/−) mice exhibited a decreased skin fibroblast proliferation rate.The heterozygous (+/−) mice exhibited an increased total tissue mass,increased fat %, and increased BMC/LBM ration, as well as increasedtrabecular bone measurements. Gene disruption was confirmed by Southernblot.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(b) Pathology

Microscopic Observations: Not tested due to embryonic lethality. At 12.5days, 40 embryos were observed: 28 (+/−) embryos, 6 (+/+) embryos, 4resorption moles, and 2 inconclusive.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

(c) Oncology Phenotypic Analysis

In the area of oncology, targets were identified herein for thetreatment of solid tumors, lymphomas and leukemia.

Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 heterozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

The (+/−) mice exhibited a decreased mean skin fibroblast proliferationrate when compared with their gender-matched (+/+) littermates and thehistorical mean. Thus, heterozygous mutant mice demonstrated ahypo-proliferative phenotype. As suggested by these observations,antagonists of a PRO1298 polypeptide or its encoding gene would beuseful in the treatment of diseases associated with abnormal cellproliferation.

(d) Bone Metabolism & Body Diagnostics: Bone Metabolism: RadiologyPhenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 heterozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (+/−) mice exhibited increased mean total tissue mass andincreased total body fat (% and gram), and an increased BMC/LBM ratiowhen compared with their gender-matched (+/+) littermates and thehistorical means.

Micro-CT: The (+/−) mice exhibited increased trabecular bone volume,number, thickness, and connectivity density as well as mean femoralmid-shaft cortical thickness area when compared with theirgender-matched (+/+) littermates and the historical means.

In summary, the (+/−) mice exhibited increased mean total tissue massand total body fat and increased trabecular bone and femoral mid-shaftbone measurements when compared with their gender-matched (+/+)littermates. These observations suggest an obesity and/or growthdisorder type phenotype. In addition, the mutant (+/−) mice exhibited anabnormal bone development. Thus, PRO1298 polypeptides or agoniststhereof, would be useful for normal growth and bone development andwould play a role in the treatment of related growth or metabolicdisorders associated with obesity and/or bone disorders.

35.21. Generation and Analysis of Mice Comprising DNA64966-1575 (UNQ679)Gene Disruptions

In these knockout experiments, the gene encoding PRO1313 polypeptides(designated as DNA64966-1575) (UNQ679) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)175187 or Mus musculus RIKENcDNA 2810446P07 gene; protein reference: NP_(—)780396 or RIKEN cDNA2810446P07 [Mus musculus]; the human gene sequence reference:NM_(—)153354 or Homo sapiens hypothetical protein MGC33214 (MGC33214);the human protein sequence corresponds to reference: NP_(—)699185 orhypothetical protein MGC33214 [Homo sapiens].

The mouse gene of interest encodes a hypothetical membrane protein(2810446P07Rik), which is the ortholog of a human hypothetical membraneprotein (MGC33214).

MGC33214 is likely to be a membrane protein, containing seventransmembrane domains. The function of this protein is currentlyunknown. MGC33214 is most similar to other hypothetical proteins from awide variety of species, including a very large (5722 amino acids) rathypothetical protein annotated as “similar to ATP binding cassettetransporter A13.” Bioinformatic analysis suggests that the protein maybe located on the plasma membrane; however, endoplasmic reticulum andnuclear membrane are also possible cell locations.

Genetics Information:

wt het hom Total Observed 22 41 8 71 Expected 17.75 35.5 17.75 71Chi-Sq. = 7.23 Significance = 0.02698 (hom/n) = 0.11 Avg. Litter Size =8

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 1 and 2(NCBI accession number NM_(—)175187.3).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.Due to lethality, transcript expression analysis was not performed.Disruption of the target gene was confirmed by Inverse PCR.

35.21.1. Phenotypic Analysis (for Disrupted Gene: DNA64966-1575 (UNQ679)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalmembrane protein (MGC33214) resulted in lethality of (−/−) mutants. Nonotable phenotype was observed for the (+/−) mice. All (−/−) pups weredead at the time of genotyping.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(b) Embryonic Expression Studies

In situ hybridization (ISH) studies:

Using a probe made to base pairs 969-1407 from the initiating ATG ofNM_(—)175187 (exons 10-12), specific and ubiquitous staining wasobserved in the embryonic placenta of (+/+) mice at E6.5d, E7.5d, E9.5d,E10.5d, E11.5d and E12.5D. Yet another probe showed UNQ679 stainingspecific to the dorsal midline (ubiquitous staining) (probe was made tobase pairs 1317-1888 from the initiating ATG of NM_(—)175187 (exon 12 tostart of 3′ UTR). On the other hand, UNQ679 homozygous (−/−) mice wereshown to be anemic and much smaller compared to the wild-type (+/+)control mice at E13.5d. Similar results were noted at E18.5d. Aplacental defect was notably displayed in the mutant (−/−) mice at 14.5dcompared to the wild-type embryos at E14.5d. In addition, the head andeyes of the UNQ679 (−/−) embryos at E16.5d were improperly formed.UNQ679 (−/−) mutant embryos were smaller, anemic and have malformedhearts at E15.5d. See EXAMPLE 44 for ISH protocol. Thus, UNQ679 isessential for placenta development and normal development. The observedanemia in the mutant (−/−) embryos at E13.5d was more than likely aresult of the defective placental development.

35.22. Generation and Analysis of Mice Comprising DNA68885-1678 (UNQ776)Gene Disruptions

In these knockout experiments, the gene encoding PRO1570 polypeptides(designated as DNA68885-1678) (UNQ776) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)145403 or ACCESSION:NM_(—)145403 NID: 21703805 Mus musculus Mus musculus similar toTransmembrane protease, serine 4 (Membrane-type serine protease 2)(MT-SP2) (LOC214523); protein reference: Q8VCA5 or ACCESSION: Q8VCA5NID: Mus musculus (Mouse). SIMILAR TO TRANSMEMBRANE PROTEASE, SERINE 4.MOUSESPTRNRDB; the human gene sequence reference: NM_(—)019894 orACCESSION: NM_(—)019894 NID: 15451939 Homo sapiens Homo sapienstransmembrane protease, serine 4 (TMPRSS4); the human protein sequencecorresponds to reference: Q9NRS4 or ACCESSION: Q9NRS4 NID: Homo sapiens(Human). TRANSMEMBRANE PROTEASE, SERINE 4 (EC 3.4.21.-) (MEMBRANE-TYPESERINE PROTEASE 2) (MT-SP2). HUMANSPTRNRDB.

The mouse gene of interest is Tmprss4 (transmembrane protease, serine4), ortholog of human TMPRSS4. Aliases include membrane-type serineprotease 2, MT-SP2, transmembrane serine protease 3, and TMPRSS3.

TMPRSS4 is a type II membrane protein that is likely to function as aserine protease of the chymotrypsin family. The protein contains asignal anchor and an extracellular trypsin-like serine protease domain.TMPRSS4 is overexpressed in pancreatic cancer but not in normalpancreas, suggesting that TMPRSS4 may be involved in metastasis(Wallrapp et al., Cancer Res, 60(10):2602-6 (2000); Gress et al., GenesChromosomes Cancer, 19(2):97-103 (1997)). TMPRSS4 may regulate renalsodium transport by activating the epithelial sodium channel (ENaC)expressed in collecting tubules (Vuagniaux et al., J Gen Physiol,120(2):191-201 (2002)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 24 33 14 71 Expected 17.75 35.5 17.75 71Chi-Sq. = 3.17 Significance = 0.20505 (hom/n) = 0.20 Avg. Litter Size =8Mutation Type: Homologous Recombination (standard)Coding exons 3 through 5 were targeted (NCBI accession NM_(—)145403.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except spinal cord, thymus, bone, andadipose.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.22.1. Phenotypic Analysis (for Disrupted Gene: DNA68885-16778(UNQ776)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human transmembraneprotease, serine 4 (TMPRSS4) resulted in an increased anxiety-relatedresponse in (−/−) mice. The (−/−) mice also exhibited an increased skinfibroblast proliferation rate. Mutant (−/−) mice also exhibitedincreased mean serum triglyceride levels, and increased mean serumglucose levels with an impaired glucose tolerance. An increased responseto the LPS challenge was also observed in the (−/−) mice. Genedisruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACSCalibur instrument.

Results:

The (−/−) mice exhibited an increased mean serum MCP-1 response to LPSchallenge when compared with their (+/+) littermates and the historicalmean.

Analyzed wt/het/hom: 7/4/11

In summary, the LPS endotoxin challenge results indicate that knockoutmice deficient in the gene encoding PRO1570 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In one instance, the mutant mice exhibited an increasedability to elicit an immunological response (MCP-1 production) whenchallenged with the LPS endotoxin indicating a proinflammatory response.MCP-1 contributes to the later stages of B cell activation. In addition,MCP-1 plays a critical role in inducing the actue phase response andsystemic inflammation. This finding suggests that inhibitors orantagonists to PRO1570 polypeptides would stimulate the immune systemand would find utility in the cases wherein this effect would bebeneficial to the individual such as in the case of leukemia, and othertypes of cancer, and in immunocompromised patients, such as AIDSsufferers. Accordingly, PRO1570 polypeptides or agonists thereof wouldbe useful in inhibiting the immune response and would be usefulcandidates for suppressing harmful immune responses, e.g. in the case ofgraft rejection or graft-versus-host diseases.

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning First, the field (40×40 cm) wasselected to be relatively large for a mouse, thus designed to pick upchanges in locomotor activity associated with exploration. In addition,there were 4 holes in the floor to allow for nose-poking, an activityspecifically related to exploration. Several factors were also designedto heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value. Analyzed wt/het/hom: 4/4/8

Results:

The (−/−) mice exhibited decreased median sum time-in-center and holepoke activity during open field testing when compared with theirgender-matched (+/+) littermates and the historical mean, suggesting anincreased anxiety-like response in the mutants.

As noted above, a notable difference was observed during open fieldactivity testing. The (−/−) mice exhibited a decreased median sum timein the center area when compared with their gender-matched (+/+)littermates. This type of behavior is consistent with an increasedanxiety like response. Thus, the knockout mice demonstrated a phenotypeconsistent with anxiety related disorders which are associated with mildto moderate anxiety, anxiety due to a general medical condition, and/orbipolar disorders; hyperactivity; sensory disorders;obsessive-compulsive disorders, schizophrenia or a paranoid personality.Thus, PRO1570 polypeptides or agonists thereof would be useful in thetreatment of such neurological disorders or the amelioration of thesymptoms associated with anxiety disorders.

(d) Oncology Phenotypic Analysis

In the area of oncology, targets were identified herein for thetreatment of solid tumors, lymphomas and leukemia.

Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

The (−/−) mice exhibited an increased mean skin fibroblast proliferationrate when compared with their gender-matched (+/+) littermates and thehistorical mean. Thus, homozygous mutant mice demonstrated ahyper-proliferative phenotype. As suggested by these observations,PRO1570 polypeptides or agonists thereof would be useful in decreasingabnormal cell proliferation such as tumor cell growth.

(e) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche).

Results:

The (−/−) mice exhibited increased mean serum triglyceride and glucoselevels when compared with their gender-matched (+/+) littermates and thehistorical means.

As summarized above, the (−/−) mice exhibited notably increasedtriglyceride levels when compared with their gender-matched (+/+)littermates and the historical means for the male (+/+) mice. Inaddition, the increased mean serum glucose levels suggesting diabetes.Thus, mutant mice deficient in the PRO1570 gene can serve as a model forcardiovascular disease including diabetes. PRO1570 polypeptides or itsencoding gene would be useful in regulating normal blood lipid levelssuch as triglycerides. Thus, PRO1570 polypeptides or agonists thereofwould be useful in the treatment of such cardiovascular diseases ashypertension, atherosclerosis, heart failure, stroke, various coronarydiseases, hypercholesterolemia, hypertriglyceridemia, diabetes and/orobesity.

35.23. Generation and Analysis of Mice Comprising DNA80796-2523 (UNQ870)Gene Disruptions

In these knockout experiments, the gene encoding PRO1886 polypeptides(designated as DNA80796-2523) (UNQ870) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)134593 or Mus musculushypothetical protein 4932417I16 (4932417I16); protein reference:XP_(—)134593 or similar to predicted CDS, putative protein of bilaterialorigin (41193) [Mus musculus]; the human gene sequence reference:AK025820 or Homo sapiens cDNA: FLJ22167 fis, clone HRC00584; the humanprotein sequence corresponds to reference: BAB15244 or unnamed proteinproduct [Homo sapiens].

The disrupted mouse gene encodes a hypothetical protein (4932417I16Rik),which is orthologous to human hypothetical protein FLJ22167. Aliasesinclude hypothetical protein 4932417I16 (murine).

Bioinformatic analyses of FLJ22167 indicate that it is a transmembraneprotein, possibly located in the plasma membrane or the endoplasmicreticulum. In human, some FLJ22167 transcripts overlap those of anadjacent gene (CHST5), but such overlap is not observed in mouse. Theoverlap has sometimes led to misidentification of FLJ22167 as a possibleenzyme (isoform of CHST5). No critically evaluated information suggeststhat FLJ22167 is an enzyme.

Genetics Information:

wt het hom Total Observed 29 28 0 57 Expected 14.25 28.5 14.25 57Chi-Sq. = 29.53 Significance = 0.00000 (hom/n) = 0.00 Avg. Litter Size =6

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred within the intron proceeding coding exon 1(NCBI Accession AK030046).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.Due to lethality, transcript expression analysis was not performed.Disruption of the target gene was confirmed by Inverse PCR.

35.23.1. Phenotypic Analysis (for Disrupted Gene: DNA80796-2523 (UNQ870)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalprotein (FLJ22167) resulted in lethality of (−/−) mutants. One fourth(¼) of the UNQ870 pups make it to birth. The (+/−) mice exhibitedincreased total body fat (% and gram) and increased mean total tissuemass. In addition, a reduced fibroblast proliferation was observed inthe (+/−) mice.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(b) Further Expression and Embryonic Observations

UNQ 870 is upregulated in endometrial adenocarcinoma (See EXAMPLES 41and 42 for protocol). Microscopic observations showed one third (⅓) ofthe heterozygous (+/−) mice to have hydronephrosis of the right kidney.

The UNQ870 homozygous (−/−) embryos showed heart defects at E15.5d.

Three fourths (¾) of UNQ870 homozygous (−/−) embryos are anemic and haveabnormal eyes at E14.5d. One fourth (¼) of the UNQ870 (−/−) embryos areanemic and have eyes at E15.5d but no eyelid closure. Normal eyeliddevelopment results in an eyelid primordia at E12.5d, a fused eyelidbetween 15.5d-16.5d and eyes open at P12-14d. There is a parallelbetween eyelid closure and wound healing (progressive events run inparallel). Also other factors such as FGF8 expression, neurogenin 2expression, Gli3 and Dlx2 expression, as well as EyaI expression allappear to be altered in the (−/−) mutant embryos. [FGF8 expression isreduced in UNQ870 (−/−) at E10.5d; reduced staining in the telencephalicvescicle and nasal placode in UNQ870 (−/−). Neurogenin 2 expression isreduced in UNQ870 (−/−) at E10.5d; reduced staining in telencephalon ofUNQ870 (−/−) or reduced or missing ventral staining in UNQ870 (−/−).Gli3 expression is altered in UNQ870 (−/−) at E10.5d; (reduced stainingin telencephalon, branchial arches, and optic vesicles). Dlx2 expressionis altered in UNQ870 (−/−) at E9.5d; tighter domain of staining at AERof limb bud in wild-type (+/+). EyaI expression is altered in UNQ870(−/−) at E9.5d; reduced staining in branchial arches and otic vesicles.See EXAMPLE 44 for ISH protocol.

Pax6 knockouts are similar to UNQ870 knockouts. Small eye (Sey) is asemidominant mutation in the Pax6 gene. Homozygotes (−/−) result in thecomplete lack of eyes and nasal primordia. On the basis of comparativemapping studies and on phenotypic similarities, Sey has been suggestedto be homologous to congenital aniridia (lack of iris) in humans.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (+/−) mice exhibited increased mean body weight when compared withtheir gender-matched (+/+) littermates and the historical means.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The (+/−) mice exhibited increased mean total tissue mass, percenttotal body fat, and total fat mass when compared with theirgender-matched (+/+) littermates and the historical means.

These studies suggest that mutant (+/−) non-human transgenic animalsexhibit a negative phenotype that may be associated with obesity. Thus,PRO1886 polypeptides or agonists thereof are essential for normal growthand metabolic processes and especially would be important in theprevention and/or treatment of obesity.

(d) Oncology Phenotypic Analysis

In the area of oncology, targets were identified herein for thetreatment of solid tumors, lymphomas and leukemia.

Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 heterozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type and(+/−) mutant mice. Duplicate or triplicate cultures of 0.05 millioncells were plated and allowed to grow for six days. At the end of theculture period, the number of cells present in the culture wasdetermined using a electronic particle counter.

Results:

The (+/−) mice exhibited a decreased mean skin fibroblast proliferationrate when compared with their gender-matched (+/+) littermates and thehistorical mean. Thus, heterozygous mutant mice demonstrated ahypo-proliferative phenotype. As suggested by these observations,antagonists of a PRO1886 polypeptide or its encoding gene would beuseful in decreasing abnormal cell proliferation such as tumor cellgrowth.

35.24. Generation and Analysis of Mice Comprising DNA76788-2526 (UNQ873)Gene Disruptions

In these knockout experiments, the gene encoding PRO1891 polypeptides(designated as DNA76788-2526) (UNQ873) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)033615 or ACCESSION:NM_(—)033615 NID: gi 23956237 ref NM_(—)033615.1 Mus musculus adisintegrin and metalloprotease domain 33 (Adam33); protein reference:Q923W9 or ACCESSION: Q923W9 NID: Mus musculus (Mouse). ADAM 33 PRECURSOR(EC 3.4.24.) (A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 33).MOUSESPTRNRDB; the human gene sequence reference: NM_(—)025220 orACCESSION: NM_(—)025220 NID: 18252044 Homo sapiens Homo sapiens adisintegrin and metalloproteinase domain 33 (ADAM33); the human proteinsequence corresponds to reference: Q9BZ11 or ACCESSION: Q9BZ11 NID: Homosapiens (Human). ADAM 33 PRECURSOR (EC 3.4.24.-) (A DISINTEGRIN ANDMETALLOPROTEINASE DOMAIN 33). HUMANSPTRNRDB.

The mouse gene of interest is Adam33 (a disintegrin and metalloproteasedomain 33), ortholog of human ADAM33. Aliases include Adam1,metalloprotease disintegrin, and disintegrin and reprolysinmetalloproteinase family protein.

ADAM33 is a type I integral membrane protein that likely functions as azinc metalloprotease and cell adhesion molecule. The protein consists ofa large extracellular domain, a transmembrane segment, and a shortcytoplasmic C-terminus. The large extracellular domain contains apropeptide, a zinc metalloprotease catalytic domain, a disintegrindomain, a cysteine rich region, and an EGF-like domain. The propeptideis cleaved to form the mature protein. ADAM33 is widely expressed, withparticularly high expression in placenta, lung, spleen, and veins.Expression of ADAM33 appears to be absent in liver (Yoshinaka et al.,Gene, 282(1-2):227-36 (2002); Van Eerdewegh et al., Nature,418(6896):426-30 (2002); Garlisi et al., Biochem Biophys Res Commun,301(1):35-43 (2003)). The biological role of ADAM33 and ADAM familymembers in general are not clearly known; however, they have beenimplicated in processes such as fertilization, neurogenesis, myogenesis,embryonic TGF-alpha release, and the inflammatory response (Primakoffand Myles, Trends Genet, 16(2):83-7 (2000)).

ADAM33 has been implicated in asthma and bronchial hyper responsiveness.The protease is expressed in human lung fibroblasts and bronchial smoothmuscle, which play a central role in airway remodeling. Damage to airwayepithelial cells by activated T cells leads to smooth musclehyperplasia, fibroblast proliferation, increased matrix deposition, andconversion of bronchial smooth muscle from the quiescent, contractiletype to the proliferative, synthetic type. Alterations in ADAM33activity or expression may underlie abnormalities in airway remodelingin bronchial hyper responsiveness (Van Eerdewegh et al., Nature,418(6896):426-30 (2002)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 15 48 21 84 Expected 21 42 21 84 Chi-Sq. =2.57 Significance = 0.27645 (hom/n) = 0.25 Avg. Litter Size = 9Mutation Type: Homologous Recombination (standard)Coding exons 12 through 16 were targeted (NCBI accessionNM_(—)033615.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except adipose.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.24.1. Phenotypic Analysis (for Disrupted Gene: DNA76788-2526 (UNQ873)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human a disintegrin andmetalloprotease domain 33 (ADAM33) resulted in an increased IgG1response to ovalbumin challenge in (−/−) mice. The (−/−) mice exhibitedincreased serum triglycerides, increased mean serum insulin anddecreased bone density measurements. In addition, glucose tolerancetesting suggested insulin resistance. Immunological abnormalities werealso observed in the (−/−) mice. Gene disruption was confirmed bySouthern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmunodominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample. Analyzed wt/het/hom:9/4/14

Results of this challenge: The (−/−) mice exhibited an increased meanserum IgG1 response to the ovalbumin challenge when compared with their(+/+) littermates. Thus, these knockout mice exhibited an increasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen. Inhibitors (antagonists) of PRO1891 polypeptideswould be expected to also stimulate the immune system and would findutility in the cases wherein this effect would be beneficial to theindividual such as in the case of leukemia, and other types of cancer,and in immunocompromised patients, such as AIDS sufferers. Accordingly,PRO1891 polypeptides or agonists thereof would be useful in inhibitingthe immune response and would be useful candidates for suppressingharmful immune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

(c) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: Both the male and female (−/−) mice exhibited decreased mean bonemineral content, bone mineral density, and volumetric bone mineraldensity when compared with their gender-matched (+/+) littermates andthe historical means.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density when comparedwith their gender-matched (+/+) littermate and the historical means.

Analyzed wt/het/hom: 4/4/8

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO1891 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO1891 or its encoding gene would be usefulin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO1891 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis, and osteopenia.

(d) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche).

Results:

The (−/−) mice exhibited an increased mean serum triglyceride level whencompared with their gender-matched (+/+) littermates and the historicalmean.

As summarized above, the (−/−) mice exhibited notably increasedtriglyceride levels when compared with their gender-matched (+/+)littermates and the historical means for the male (+/+) mice. Thus,mutant mice deficient in the PRO1891 gene can serve as a model forcardiovascular disease. PRO1891 polypeptides or its encoding gene wouldbe useful in regulating blood lipids such as triglycerides. Thus,PRO1891 polypeptides or agonists thereof would be useful in thetreatment of such cardiovascular diseases as hypertension,atherosclerosis, heart failure, stroke, various coronary diseases,hypercholesterolemia, hypertriglyceridemia, diabetes and/or obesity.

(e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/hom: 4/4/8

Insulin Data:

Test Description Lexicon Genetics uses the Cobra II Series Auto-GammaCounting System in its clinical settings for running quantitativeInsulin assays on mice.

Results:

The (−/−) mice exhibited an increased mean serum insulin level whencompared with their gender-matched (+/+) littermates and the historicalmean.

However, blood chemistry testing showed (−/−) mice exhibiting anincreased mean serum glucose level when compared with theirgender-matched (+/+) littermates and the historical mean. During theglucose tolerance test, the (−/−) mice exhibited an increased meanfasting serum glucose level when compared with their gender-matched(+/+) littermates and the historical mean.

Thus, knockout mice exhibited the phenotypic pattern of an impairedglucose homeostasis with elevated levels of fasting serum glucoseindicative of diabetes or a pre-diabetic condition even in the presenceof increased insulin levels. Based on these results, PRO1891 (oragonists thereof) or its encoding gene would be useful in the treatmentof an impaired glucose metabolism (marked by insulin resistance) and/ordiabetes.

35.25. Generation and Analysis of Mice Comprising DNA88004-2575(UNQ1934) Gene Disruptions

In these knockout experiments, the gene encoding PRO4409 polypeptides(designated as DNA88004-2575) (UNQ1934) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)028065 or ACCESSION:NM_(—)028065 NID: gi 21312509 ref NM_(—)028065.1 Mus musculus RIKEN cDNA1600025D17 gene (1600025D17Rik); protein reference: Q9DAU1 or ACCESSION:Q9DAU1 NID: Mus musculus (Mouse). 1600025D17Rik protein (Putativeretinoic acid-regulated protein) (RIKEN cDNA 1600025D17 gene).MOUSESPTRNRDB; the human gene sequence reference: NM_(—)006586 or Homosapiens trinucleotide repeat containing 5 (TNRC5); the human proteinsequence corresponds to reference: Q9BT09 or ACCESSION: Q9BT09 NID: Homosapiens (Human). Hypothetical protein (DJ475N16.1) (CTG4A).HUMANSPTRNRDB.

The mouse gene of interest encodes a hypothetical protein(1600025D17Rik), which is the ortholog of human protein TNRC5(trinucleotide repeat containing 5). Aliases include CAG repeatcontaining expanded repeat domain and CAG/CTG 5.

TNRC5 is a hypothetical protein containing CAG repeats, which are likelyto be involved in specific diseases, most with neuropsychiatric features(Margolis et al., Hum Genet, 100(1):114-22 (1997)). The protein containsa signal peptide but no other identifiable domains. The cell location ofTNRC5 is ambiguous; bioinformatic analysis suggests that the protein maybe secreted or located in the endoplasmic reticulum. TNRC5 genetranscription is likely to be under the control of retinoids, which playan important role in development and physiology (Glozak et al., MolEndocrinol, 17(1):27-41 (2003)).

Genetics Information:

wt het hom Total Observed 20 37 10 67 Expected 16.75 33.5 16.75 67Chi-Sq. = 3.72 Significance = 0.15595 (hom/n) = 0.15 Avg. Litter Size =8

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 1 and 2(NCBI accession NM_(—)028065.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and, among the 13 adult tissue samples tested by RT-PCR, inkidney; stomach, small intestine, and colon; and adipose.Due to reduced viability, transcript expression analysis was notperformed. Disruption of the target gene was confirmed by Inverse PCR.

35.25.1. Phenotypic Analysis (for Disrupted Gene: DNA88004-2575(UNQ1934)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human trinucleotide repeatcontaining 5 (TNRC5) resulted in small (−/−) mice that failed to thrive.UNQ1934 was shown to be ubiquitously expressed between 7.5d-12.5d ofembryo development in wild-type pups. The (−/−) mutants were euthanizedor transferred to necropsy by 3 weeks of age. 40% fewer than expectedhomozygotes were present at genotyping. Heterozygous (+/−) miceexhibited decreased total tissue mass and fat (% and gram) compared totheir wild-type littermate controls and the historical mean.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(b) Body Diagnostics—Tissue Mass & Lean Body Mass Measurements—Dexa

(1) Dexa Analysis—Test Description:

Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

General Observations: The (−/−) mice were small, failed to thrive, andwere euthanized or sent to pathology for analysis by 3 weeks of age.

Weight: The (−/−) mice exhibited decreased mean body weight at the 2week measurement when compared with their gender-matched (+/+)littermates and the historical means.

Pathology:

Microscopic Observations: Though the (−/−) mice were smaller than their(+/+) littermates and died before 4 weeks of age, no histopathologicallesions were observed that explained the early mortality.

Gene Expression: Expression of the neo transcript was not detected inthe panel of tissues analyzed by in situ hybridization.

Summary

The (−/−) mice analyzed exhibited notably decreased viability shown by afailure to survive past four weeks of age when compared with their (+/+)littermates. The (−/−) mice were quite small in size and showed anotable decrease in body weight suggestive of growth retardation inthese mutants. Although pathology observations failed to reveal anyhistopathological lesions, the negative phenotype is indicative of atissue wasting condition with severe growth retardation. Thus, PRO4409polypeptides or agonists thereof must be essential for normal growthand/or growth metabolism and therefore would be useful in the treatmentor prevention of growth disorders such as cachexia or other tissuewasting diseases.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The (+/−) mice exhibited decreased mean total tissue mass, percenttotal body fat, and total fat mass when compared with theirgender-matched (+/+) littermates and the historical means.

These studies suggest that mutant (+/−) non-human transgenic animalsexhibit a negative phenotype that may be associated with tissue wastingdiseases. Thus, PRO4409 polypeptides or agonists thereof are essentialfor normal growth and metabolic processes and especially would beimportant in the prevention and/or treatment of growth disorders and/ortissue wasting diseases.

35.26. Generation and Analysis of Mice Comprising DNA92265-2669(UNQ2446) Gene Disruptions

In these knockout experiments, the gene encoding PRO5725 polypeptides(designated as DNA92265-2669) (UNQ2446) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)175024 or Mus musculus RIKENcDNA G630049C14 gene (G630049C14Rik); protein reference: Q8BN06 orACCESSION: Q8BN06 NID: Mus musculus (Mouse). Hypothetical protein; thehuman gene sequence reference: NM_(—)198443 or Homo sapiens MRCC2446(UNQ2446); the human protein sequence corresponds to reference:NP_(—)940845 or MRCC2446 [Homo sapiens] gi|37182683|gb|AAQ89142.1|MRCC2446 [Homo sapiens].

The targeted mouse gene encodes a hypothetical protein (G630049C14Rik),which is the ortholog of hypothetical human protein MRCC2446. The humangene is also known as UNQ2446.

The human and mouse genes encode putative secreted proteins. The mousehypothetical protein contains a signal peptide, a C-terminaltransmembrane segment, and no other identifiable domains. The humanprotein contains a signal peptide and no other identifiable domains.Both proteins appear to be distantly similar to neuritin, anextracellular protein anchored on the plasma membrane of neurons that islikely to be involved in neuritogenesis (Naeve et al., Proc Natl AcadSci USA, 94(6):2648-53 (1997)).

Genetics Information:

wt het hom Total Observed 20 30 15 65 Expected 16.25 32.5 16.25 65Chi-Sq. = 1.15 Significance = 0.56162 (hom/n) = 0.23 Avg. Litter Size =7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in coding exon 2 (Accession:NM_(—)175024.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptskeletal muscle.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-121).

35.26.1. Phenotypic Analysis (for Disrupted Gene: DNA92265-2669(UNQ2446)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a hypothetical humanprotein resulted in the observation of decreased body weight, decreasedlean body mass, bone mineral density and bone mineral content as well asdecreased lumbar 5 measurements. Transcript was absent by RT-PCR.

(b) Body Diagnostics—Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The male and female (−/−) mice exhibited decreased mean body weight whencompared with their gender-matched (+/+) littermates and the historicalmeans. Analyzed wt/het/hom: 28/36/22

The (−/−) mice were quite small in size and showed a notable decrease inbody weight suggestive of growth retardation in these mutants. Althoughpathology observations failed to reveal any histopathological lesions,the negative phenotype suggested growth retardation. Thus, PRO5725polypeptides or agonists thereof must be essential for normal growthand/or growth metabolism and therefore would be useful in the treatmentor prevention of growth related disorders.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean lean body mass, bonemineral content and bone mineral density when compared with theirgender-matched (+/+) littermates and the historical means.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connectivity density and decreasedmean femoral mid-shaft cortical thickness when compared with theirgender-matched (+/+) littermates and the historical means.

Analyzed wt/het/hom: 4/4/8

Summary

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO5725 or agonists thereof would play a role in maintaining bonehomeostasis. In addition, PRO5725 or its encoding gene would be usefulfor maintaining bone homeostasis and for bone healing or for thetreatment of arthritis or osteoporosis; whereas antagonists to PRO5725or its encoding gene would lead to abnormal or pathological bonedisorders including inflammatory diseases associated with abnormal bonemetabolism including arthritis, osteoporosis, and osteopenia.

The (−/−) mice analyzed by DEXA exhibited notably decreased lean bodymass when compared with their (+/+) littermates, suggestive of growthretardation in these mutants. This in conjunction with the observationsof abnormal bone measurements suggest a tissue wasting condition orother growth related disorders such as cachexia. Thus, PRO5725polypeptides or agonists thereof would be useful in the treatment ofbone disorders but also would be useful for the prevention of growthrelated disorders such as cachexia and/or other tissue wasting diseases.

35.27. Generation and Analysis of Mice Comprising DNA98591 (UNQ2506)Gene Disruptions

In these knockout experiments, the gene encoding PRO5994 polypeptides(designated as DNA98591 (UNQ2506) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)011463 or Mus musculus serineprotease inhibitor, Kazal type 4 (Spink4); protein reference: 035679 orACCESSION: O35679 NID: Mus musculus (Mouse). SERINE PROTEASE INHIBITORKAZAL-TYPE 4 PRECURSOR (PEPTIDE PEC-60 HOMOLOG) (MPGC60 PROTEIN).MOUSESPTRNRDB; the human gene sequence reference: NM_(—)014471 orACCESSION: NM_(—)014471 NID: gi 7657452 ref NM_(—)014471.1 Homo sapiensserine protease inhibitor, Kazal type 4 (SPINK4); the human proteinsequence corresponds to reference: O60575 or ACCESSION: O60575 NID: Homosapiens (Human). SERINE PROTEASE INHIBITOR KAZAL-TYPE 4 PRECURSOR(PEPTIDE PEC-60 HOMOLOG). HUMANSPTRNRDB.

The mouse gene of interest is Spink4 (serine protease inhibitor, Kazaltype 4), ortholog of human SPINK4. Aliases include MPGC60, PEC-60, andgastrointestinal peptide.

SPINK4 is a putative secreted serine protease inhibitor expressedpredominantly in the intestinal tract (Krause et al., Differentiation,63(5):285-94 (1998)). SPINK4 is the apparent human ortholog of porcinePEC-60 (SwissProt P37109), which has been shown to inhibitglucose-induced insulin secretion. PEC-60 has been isolated from brainsof rat and pig (Norberg et al., Cell Mol Life Sci, 60(2):378-81 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 21 41 27 89 Expected 22.25 44.5 22.25 89Chi-Sq. = 1.36 Significance = 0.50673 (hom/n) = 0.30 Avg. Litter Size =9Mutation Type: Homologous Recombination (standard)Coding exon 1 was targeted (NCBI accession NM_(—)011463.1).Wild-type expression of the target gene was detected in all 13 adulttissue samples tested by RT-PCR, except brain, lung, and skeletalmuscle.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.27.1. Phenotypic Analysis (for Disrupted Gene: DNA98591 (UNQ2506)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human serine proteaseinhibitor, Kazal type 4 (SPINK4) resulted in decreased body weight andlength as well as decreased tissue mass measurements. In addition, the(−/−) mice exhibit lymphoid hyperplasia and tissue inflammation at anincreased incidence. Gene disruption was confirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

General Observations: The agouti (−/−) mouse is much smaller than itsblack (+/+) littermate.

The (−/−) mice exhibited decreased mean body weight and decreased bodylength when compared with the historical means.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The (−/−) mice exhibited decreased mean total tissue mass and leanbody mass when compared with their gender-matched (+/+) littermates andthe historical means.

Analyzed wt/het/hom: 4/4/8

Summary

These results demonstrate that the knockout mutant (−/−) mice analyzedby DEXA were quite small in size and exhibited a decrease in body weightand length as well as a decrease in total tissue, lean body masssuggestive of growth retardation in these mutants. Thus, PRO5994polypeptides or agonists thereof must be essential for normal growthand/or growth metabolism and therefore would be useful in the treatmentor prevention of growth disorders, cachexia or other tissue wastingdiseases.

(c) Pathological Observations

The knockout (−/−) mice exhibited lymphoid hyperplasia and tissueinflammation at an increased incidence.

35.28. Generation and Analysis of Mice Comprising DNA107701-2711(UNQ2545) Gene Disruptions

In these knockout experiments, the gene encoding PRO6097 polypeptides(designated as DNA107701-2711) (UNQ2545) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)028787 or Mus musculussolute carrier family 35, member F5 (Slc35f5); protein reference: Q9DBK9or Q9DBK9 Q9DBK9 1300003P13RIK PROTEIN; the human gene sequencereference: NM_(—)025181 or Homo sapiens solute carrier family 35, memberF5 (SLC35F5); the human protein sequence corresponds to reference:Q8WV83 or ACCESSION: Q8WV83 NID: Homo sapiens (Human). Similar to RIKENcDNA 1300003P13 gene (NS5ATP3).

The mouse gene of interest is Slc35f5 (solute carrier family 35, memberF5), ortholog of human SLC35F5. Aliases include 1300003P13Rik andFLJ22004.

SLC35F5 is a predicted integral plasma membrane protein of unknownfunction, containing 10 transmembrane segments. Most of thetransmembrane segments are contained within a domain that has weaksimilarity to that found in carbohydrate/phosphate translocators (KOG4313) and drug permeases (COG0697) (Marchler-Bauer et al., Nucleic AcidsRes, 31 (1): 383-7 (2003).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 17 35 12 64 Expected 16 32 16 64 Chi-Sq. =1.34 Significance = 0.51075 (hom/n) = 0.19 Avg. Litter Size = 7Mutation Type: Homologous Recombination (standard)Coding exons 1 through 3 were targeted (NCBI accession NM_(—)028787.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptbone.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.28.1. Phenotypic Analysis (for Disrupted Gene: DNA107701-2711(UNQ2545)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human solute carrierfamily 35, member F5 (SLC35F5) resulted in larger (−/−) mice, exhibitingincreased body weight, total tissue mass, lean body mass and bonemeasurements. In addition, the mutant knockout mice exhibited adecreased or reduced IgG2a response to an ovalbumin challenge. Also,additional immunological abnormalities were exhibited by observing anincreased percentage of T cells, with decreased natural killer cells andB cells and an increased percentage of CD4 T cells in the mutant (−/−)mice compared with their littermate controls. Gene disruption wasconfirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmunodominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Analyzed wt/het/hom: 8/4/9

Results of this Challenge:

The (−/−) mice exhibited a decreased mean serum IgG2a response toovalbumin challenge when compared with their (+/+) littermates and thehistorical means. Thus, these knockout mice exhibited a decreasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO6097 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited a decreasedability to elicit an immunological response when challenged with theT-cell dependent OVA antigen. Thus, PRO6097 polypeptides or agoniststhereof, would be useful for stimulating the immune system (such as Tcell proliferation) and would find utility in the cases wherein thiseffect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immunocompromised patients,such as AIDS sufferers. Accordingly, inhibitors (antagonists) of PRO6097polypeptides would be useful for inhibiting the immune response and thuswould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

Flourescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio. The mononuclear cell profile was derived by staining asingle sample of lysed peripheral blood from each mouse with a panel ofsix lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

FACS: The (−/−) mice exhibited an increased percentage of T cells(increased % of CD4 T cells) with a decreased mean percentage of naturalkiller cells and B cells when compared with their (+/+) littermates andthe historical mean. Analyzed wt/het/hom: 7/4/8

In summary, the FACS results indicate that the homozygous mutant micedemonstrate immunological abnormalities marked by increased T cellpopulation but a decreased mean percentage of natural killer cells and Bcells. [Natural killer cells are the first line of defense to viralinfection since these cells have been implicated in viral immunity andin defense against tumors]. Natural killer cells or NK cells act aseffectors in antibody-dependent cell-mediated cytotoxicity and have beenidentified by their ability to kill certain lymphoid tumor cell lines invitro without the need for prior immunization or activation. However,their known function in host defense is in the early phases of infectionwith several intracellular pathogens, particularly herpes viruses]. Onthe otherhand, by knocking out the gene which encodes PRO6097polypeptides a beneficial effect is shown by the increase in the T cellpopulation. Thus, PRO6097 polypeptides or the gene encoding PRO6097appears to act as a negative regulator of T cell proliferation. Anopposite effect was shown for the B cell and natural killer cellpopulation.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited increased mean body weight when compared withtheir gender-matched (+/+) littermates and the historical mean. Analyzedwt/het/hom: 21/40/15

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited increased mean total tissue mass, leanbody mass, bone mineral content, and bone mineral density in total bodyand femur when compared with their gender-matched (+/+) littermates andthe historical means. Analyzed wt/het/hom: 4/4/8

MicroCT: The knockouts (−/−) exhibited an increased trabecular bonevolume, number, and connectivity density compared with their littermatecontrols.

In summary, the (−/−) mice exhibited increased body weight and length,increased mean total tissue mass and lean body mass and increased bonemineral density measurements and trabecular bone measurements whencompared with their gender-matched (+/+) littermates. The heterozygousmice (+/−) exhibited similar bone effects but the increased measurementvalues were between the wild-type and homozygous values. Theseobservations suggest an obesity and/or growth disorder type phenotype.In addition, the mutant (−/−) mice exhibited abnormal bone measurements.Thus, PRO6097 polypeptides or agonists thereof, would be useful fornormal growth and bone development and would play a role in thetreatment of related growth or metabolic disorders associated withobesity and/or bone disorders such as osteopetrosis.

35.29. Generation and Analysis of Mice Comprising DNA108792-2753(UNQ2966) Gene Disruptions

In these knockout experiments, the gene encoding PRO7425 polypeptides(designated as DNA108792-2753) (UNQ2966) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)176993 or Mus musculusRIKEN cDNA A530065I17 gene (A530065I17Rik); protein reference:NP_(—)795967 or RIKEN cDNA A530065I17 gene [Mus musculus]; the humangene sequence reference: NM_(—)198275 or Homo sapiens hypotheticalprotein LOC196264 (LOC196264); the human protein sequence corresponds toreference: NP_(—)938016 or hypothetical protein LOC196264 [Homosapiens].

The disrupted mouse gene is a hypothetical protein (interim name,A530065I17Rik), which is the ortholog of human hypothetical proteinFLJ38080. Aliases for the human locus include LOC196264 and QQRG2966.

Analysis of FLJ38080 indicates that two transmembrane domains (1 neareach end) flank a central Igv-type motif (InterPro IPR0003596). Suchfeatures suggest the protein may be partially or completelyextracellular, and perhaps participates in signaling or protein-proteininteractions.

Genetics Information:

wt het hom Total Observed 16 32 20 68 Expected 17 34 17 68 Chi-Sq. =0.71 Significance = 0.70262 (hom/n) = 0.29 Avg. Litter Size = 8

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 2 and 3(NCBI accession BQ713326).Wild-type expression of the target gene was detected in all 13 adulttissues samples tested by RT-PCR, except thymus, lung, skeletal muscleand adipose.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-75).

35.29.1. Phenotypic Analysis (for Disrupted Gene: DNA108792-7425(UNQ2966)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalprotein (FLJ38080) resulted in sebaceous gland hyperplasia and severegrowth retardation in (−/−) mice. In addition, the (−/−) mice exhibiteddecreased mean body mass-related measurements and decreased bonemeasurements. The (−/−) mice also exhibited severe depletion of body fatdepots and enlarged kidneys. Lower insulin levels were also observed inthe (−/−) mice as well as abnormal changes in alkaline phosphatase (mostlikely due to decreased bone mineralization) and mean serum ALT (alanineamino transaminase) suggestive of liver disease. Furthermore, the mutant(−/−) mice exhibited severe hypoactivity in the circadian rhythm test.Transcript was absent by RT-PCR.

(b) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning First, the field (40×40 cm) wasselected to be relatively large for a mouse, thus designed to pick upchanges in locomotor activity associated with exploration. In addition,there were 4 holes in the floor to allow for nose-poking, an activityspecifically related to exploration. Several factors were also designedto heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

The (−/−) mice exhibited a decreased normalized slope during open fieldactivity testing when compared with their (+/+) littermates, suggestingan abnormal habituation response to a novel environment in the mutants.

Circadian Test Description:

Female mice are individually housed at 4 pm on the first day of testingin 48.2 cm×26.5 cm home cages and administered food and water adlibitum. Animals are exposed to a 12-hour light/dark cycle with lightsturning on at 7 am and turning off at 7 pm. The system software recordsthe number of beam interruptions caused by the animal's movements, withbeam breaks automatically divided into ambulations. Activity is recordedin 60, one-hour intervals during the three-day test. Data generated aredisplayed by median activity levels recorded for each hour (circadianrhythm) and median total activity during each light/dark cycle(locomotor activity) over the three-day testing period. Thus, the mutant(−/−) mice exhibited severe hypoactivity. Analyzed wt/het/hom: 4/4/8

Results:

The (−/−) mice exhibited abnormal activity during the 1-hourhabituation, the 12-hour habituation, and all light and dark periods ofhome cage activity testing when compared with their gender-matched (+/+)littermates and the historical means. The (−/−) mice exhibited nodiurnal alternations during the 3-day testing period.

As summarized above, notable differences were observed during home-cageactivity testing. The (−/−) mice exhibited decreased ambulatory countsduring the day 2 light period when compared with their (+/+)littermates. In addition, the homozygous (−/−) mice exhibited decreasedlight-to-dark and light-to-total activity ratios when compared withtheir (+/+) littermates, suggesting an abnormal circadian rhythmsresponse in the mutants. These results are consistent with the findingsduring open field testing and indicate that the homozygous mutant miceexhibit circadian rhythms which are usually associated with lethargy ordepressive disorders. Thus, PRO7425 polypeptides or its encoded genewould be useful in the treatment of such neurological disordersincluding depressive disorders or other decreased anxiety-like symptoms.

Functional Observational Battery (FOB) Test

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Results:

Basic Sensory & Motor Observations: All 8 (−/−) mice exhibited thinningfur with bald patches during the functional observational batterytesting when compared with their (+/+) littermates.

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

General Observations: The (−/−) mice are much smaller with oily thinningfur with bald patches and exhibited darker, stickier feces than their(+/+) littermates.

Both the male and female (−/−) mice exhibited decreased mean body weightand decreased mean body length when compared with their gender-matched(+/+) littermates and the historical means.

The (−/−) mice also exhibited a decreased mean heart rate when comparedwith their gender-matched (+/+) littermates and the historical mean.

Analyzed wt/het/hom: 12/23/17

Pathology:

Microscopic Observations The (−/−) mice exhibited diffuse, but enhancedsebaceous gland hyperplasia. The skin lesions in the mutants varied inseverity from one area to another and were generally not associated withinflammation. The majority of sebaceous glands were active andhyperplastic, even in areas containing anagenic hair follicles.

Gene Expression: Expression of the neo transcript was not detected inthe panel of tissues analyzed by in situ hybridization. Analyzedwt/het/hom: 2/1/6

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

CAT-Scan Protocol:

Mice were injected with a CT contrast agent, Omnipaque 300 (NycomedAmershan, 300 mg of iodine per ml, 0.25 ml per animal, or 2.50-3.75 giodine/kg of body weight) intraperitoneally. After resting in the cagefor ˜10 minutes, the mouse was then sedated by intraperitoneal injectionof Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight). ACAT-scan was performed using a MicroCAT scanner (ImTek, Inc.) with theanesthetized animal lying prone on the test bed. Three dimensionalimages were reconstructed by the Feldkamp algorithm in a cluster ofworkstations using an ImTek 3D RECON software.

Results:

DEXA: Both the male and female (−/−) mice exhibited decreased mean bodymass-related measurements (total tissue mass, lean body mass, total fatmass, and percent total body fat) and decreased mean bonemineral-related measurements (bone mineral content, volumetric bonemineral density, and bone mineral density in total body, vertebrae, andfemur) when compared with their gender-matched (+/+) littermates and thehistorical means, suggesting severe growth retardation in the mutants.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connective density and decreasedmean femoral mid-shaft cortical thickness and cross-sectional area whencompared with their gender-matched (+/+) littermates and the historicalmeans.

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO7425 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO7425 or its encoding gene would be usefulin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO7425 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis, and osteopenia.

CAT-Scan: All 3 (−/−) mice available for testing exhibited generallydecreased body size and severe depletion of abdominal and subcutaneousbody fat depots. Bilaterally enlarged kidneys were noted in 2 mutants(M-83 and M-121) when compared with their (+/+) and (+/−) littermates ofnormal body size. However, no obvious lesion was observed in thekidneys. Analyzed wt/het/hom: 4/4/9

Thus in summary, the (−/−) mice analyzed by DEXA were quite small insize and exhibited a notable decrease in body weight and length as wellas a notable depletion of body fat depots, decrease in total tissue,lean body mass and decreased bone mineral content and density suggestiveof growth retardation in these mutants. These observations areconsistent with a tissue wasting condition and/or growth retardation.Thus, PRO7425 polypeptides or agonists thereof must be essential fornormal growth and/or growth metabolism and therefore would be useful inthe treatment or prevention of growth disorders, cachexia or othertissue wasting diseases.

(d) Phenotypic Analysis: Metabolism-Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes measuringserum insulin levels as an indicator of changes in glucose metabolism.Abnormal glucose metabolism can be related to the following disorders orconditions: Diabetes Type 1 and Type 2, Syndrome X, variouscardiovascular diseases and/or obesity.

Insulin Data:

Test Description: Lexicon Genetics uses the Cobra II Series Auto-GammaCounting System in its clinical settings for running quantitativeInsulin assays on mice.

Results:

The (−/−) mice exhibited a decreased mean serum insulin level whencompared with their gender-matched (+/+) littermates and the historicalmean. In addition, other Blood Chemistry analysis indicated that themutant (−/−) mice showed increased serum alkaline phosphatase and meanserum ALT (alanine amino transaminase) suggestive of liver disease.Ketones were also noted in the urine of the mutant mice. These resultsindicate a diabetic phenotype.

35.30. Generation and Analysis of Mice Comprising DNA129542-2808(UNQ3103) Gene Disruptions

In these knockout experiments, the gene encoding PRO10102 polypeptides(designated as DNA129542-2808) (UNQ3103) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)021319 or Mus musculuspeptidoglycan recognition protein-like (Pglyrpl-pending); proteinreference: NP_(—)067294 or peptidoglycan recognition protein-like;TAG-like [Mus musculus]; the human gene sequence reference: NM_(—)052890or Homo sapiens peptidoglycan recognition protein L precursor (PGRP-L);the human protein sequence corresponds to reference: NP_(—)443122 orpeptidoglycan recognition protein L precursor [Homo sapiens].

The targeted mouse gene is peptidoglycan recognition protein-like(Pglyrpl-pending), ortholog of human peptidoglycan recognition protein Lprecursor (PGRP-L). Aliases include TAGL, tagL, PGRP-L, TAGL-beta,tagl-beta, TAGL-alpha, tagL-alpha, PGLYRPL, TAGL-like, and TAG-like.

PGRP-L is an evolutionarily conserved N-acetylmuramoyl-L-alanine amidaseexpressed in liver that cleaves the lactylamide bond in bacterial cellwall peptidoglycan (Gelius et al., Biochem Biophys Res Commun,306(4):988-94 (2003)). Bioinformatic analysis of PGRP-L suggests thatthe protein is secreted, containing a signal peptide and a C-terminal“N-acetylmuramoyl-L-alanine amidase” domain (Pfam 01510). However,PGRP-L may be an integral membrane protein (Kibardin et al., J Mol Biol,326(2):467-74 (2003)).

Variants lacking the C-terminal amidase catalytic domain are still ableto bind with gram-positive bacteria, gram-negative bacteria, andpeptidoglycan, indicating that the catalytic and peptidoglycan-bindingdomains are separate (Kibardin et al, 2003). PGRP-L is likely to play arole in innate immunity, recognizing and degrading bacteria in liver(Gelius et al., Biochem Biophys Res Commun, 306(4):988-94 (2003);Kibardin et al., J Mol Biol, 326(2):467-74 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 17 35 16 68 Expected 17 34 17 68 Chi-Sq. =0.09 Significance = 0.95684 (hom/n) = 0.24 Avg. Litter Size = 7Mutation Type: Homologous Recombination (standard)Exons 1 and 2 were targeted (NCBI NM_(—)021319).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR, exceptkidney, testis, and bone.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

35.30.1. Phenotypic Analysis (for Disrupted Gene: DNA129542-2808(UNQ3103)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human peptidoglycanrecognition protein L precursor (PGRP-L) resulted in a decreasedanxiety-related response in female (−/−) mice. However, the male (−/−)mice did not exhibit the same response. There was a strong trend towardsstress-induced hyperthermia noted for the mutant (−/−) mice. Theseobservations show an anxiety-related phenotype but a liver specificexpression pattern does not suggest these behavioral effects. Genedisruption was confirmed by Southern blot.

(b) Expression Pattern

UNQ3103 shows a very specific expression in the liver (see EXAMPLE 41for protocol)

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning First, the field (40×40 cm) wasselected to be relatively large for a mouse, thus designed to pick upchanges in locomotor activity associated with exploration. In addition,there were 4 holes in the floor to allow for nose-poking, an activityspecifically related to exploration. Several factors were also designedto heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

A difference was found during open field testing. The female (−/−) miceexhibited an increased median sum time in center when compared withtheir gender-matched (+/+) littermates and the historical mean,suggesting a decreased anxiety-like response in the mutants. Male (−/−)mice did not exhibit the same response.

As noted above, a difference was observed during open field activitytesting. The female (−/−) mice exhibited an increased median sum time inthe center area when compared with their gender-matched (+/+)littermates, which is indicative of a decreased anxiety-like response inthe mutants. Thus, knockout mice demonstrated a phenotype consistentwith depressive disorders, schizophrenia and/or bipolar disorders. Thus,PRO10102 polypeptides and agonists thereof would be useful for thetreatment or amelioration of the symptoms associated with depressivedisorders.

Functional Observational Battery (FOB) Test

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Results:

The mutant (−/−) mice showed a strong trend towards increased stressinduced hyperthermia (three-fourths 3/4 (−/−) mice tested showedincreases two standard deviations (2 SD) above the historical mean).

35.31. Generation and Analysis of Mice Comprising DNA148380-2827(UNQ3126) Gene Disruptions

In these knockout experiments, the gene encoding PRO10282 polypeptides(designated as DNA148380-2827) (UNQ3126) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)009291 or ACCESSION:NM_(—)009291 NID: gi 6678170 ref NM_(—)009291.1 Mus musculus stimulatedby retinoic acid gene 6 (Stra6); protein reference: 070491 or ACCESSION:O70491 NID: Mus musculus (Mouse). Retinoic acid-responsive protein.MOUSESPTRNRDB; the human gene sequence reference: NM_(—)022369 orACCESSION: NM_(—)022369 NID: gi 21314699 ref NM_(—)022369.2 Homo sapienshypothetical protein FLJ12541 similar to Stra6 (FLJ12541); the humanprotein sequence corresponds to reference: Q9BX79 or ACCESSION: Q9BX79NID: Homo sapiens (Human). STRA6 isoform 1. HUMANSPTRNRDB.

The mouse gene of interest is Stra6 (stimulated by retinoic acid gene6), ortholog of human “stimulated by retinoic acid gene 6.” Aliasesinclude FLJ12541.

Stra6 is a likely integral plasma membrane protein induced by retinoicacid. The protein contains eight transmembrane segments but no otherdiscernable domain. Stra6 is often located at blood-organ barriers andmay function as a component of a transport apparatus. Stra6 is expressedin Sertoli cells during specific stages of spermatocyte development(Bouillet et al., Mech Dev, 63(2):173-86 (1997)) and in developing mouselimbs (Chazaud et al., Dev Genet, 19(1):66-73 (1996)). Wnt-1 potentiatesretinoid-induced Stra6 expression, and Stra6 mRNA may be overexpressedin human colorectal cancers as well as other tumors driven by activationof the Wnt-1/beta-catenin pathway (Szeto et al., Cancer Res,61(10):4197-205 (2001); Tice et al., J Biol Chem, 277(16):14329-35(2002)).

Genetics Information:

wt het hom Total Observed 16 34 19 69 Expected 17.25 34.5 17.25 69Chi-Sq. = 0.28 Significance = 0.87138 (hom/n) = 0.28 Avg. Litter Size =7

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron between coding exons 1 and 2(NCBI accession NM_(—)009291.1).Wild-type expression of the target gene was detected in brain, eye,thymus, and lung among the 13 adult tissue samples tested by RT-PCR.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-76). Disruption of the target gene was confirmed byInverse PCR.

35.31.1. Phenotypic Analysis (for Disrupted Gene: DNA148380-2827(UNQ3126)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human putative “stimulatedby retinoic acid gene 6” (STRA6) resulted in growth retardation anddecreased bone measurements in (−/−) mice. An increased level of meanserum glucose was also observed in (−/−) mice. Transcript was absent byRT-PCR.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

The (−/−) mice exhibited decreased mean body weight and decreased meanbody weight when compared with their gender-matched (+/+) littermatesand the historical means. Analyzed wt/het/hom: 16/37/19

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited decreased mean total tissue mass and leanbody mass when compared with their gender-matched (+/+) littermates andthe historical means. In addition to these changes noted, fat (% andgram), bone mineral density and bone mineral content were all lowerrelative to their wild-type littermates consistent with the decreasedsize of the (−/−) mice.

Micro-CT: The (−/−) mice exhibited decreased mean vertebral trabecularbone volume, number, thickness, and connective density and decreasedmean femoral mid-shaft cortical thickness and cross-sectional area whencompared with their gender-matched (+/+) littermates and the historicalmeans.

These results demonstrate that knockout mutant mice exhibit abnormalbone metabolism with significant bone loss similar to osteoporosischaracterized by decrease in bone mass with decreased density andpossibly fragility leading to bone fractures. Thus, it appears thatPRO10282 or agonists thereof would be useful in maintaining bonehomeostasis. In addition, PRO10282 or its encoding gene would be usefulin bone healing or for the treatment of arthritis or osteoporosis;whereas antagonists to PRO10282 or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis, and osteopenia.

Thus in summary, the (−/−) mice analyzed by DEXA were quite small insize and exhibited a notable decrease in body weight and length as wellas a notable depletion of body fat depots, decrease in total tissuemass, lean body mass and decreased bone mineral content and densitysuggestive of growth retardation in these mutants. Thus, PRO10282polypeptides or agonists thereof must be essential for normal growthand/or growth metabolism and therefore would be useful in the treatmentor prevention of growth disorders, cachexia or other tissue wastingdiseases.

(c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection. Analyzedwt/het/hom: 4/4/8

Results:

Blood Chemistry The (−/−) mice exhibited an increased mean serum glucoselevel when compared with their gender-matched (+/+) littermates and thehistorical mean. During the glucose tolerance test, the (−/−) miceexhibited an increased mean fasting serum glucose level when comparedwith their gender-matched (+/+) littermates and the historical mean.

Thus, knockout mice exhibited the phenotypic pattern of an impairedglucose homeostasis with elevated levels of fasting serum glucoseindicative of diabetes or a pre-diabetic condition. Based on theseresults, PRO10282 (or agonists thereof) or its encoding gene would beuseful in the treatment of an impaired glucose metabolism and/ordiabetes.

35.32. Generation and Analysis of Mice Comprising DNA347767 (UNQ14964)Gene Disruptions

In these knockout experiments, the gene encoding PRO61709 polypeptides(designated as DNA347767 (UNQ14964) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)172294 or Mus musculussulfatase 1 (Sulf1); protein reference: Q8K007 or ACCESSION: Q8K007 NID:Mus musculus (Mouse). Extracellular sulfatase Sulf-1 precursor (EC3.1.6.-) (MSulf-1); the human gene sequence reference: NM_(—)015170 orHomo sapiens sulfatase 1 (SULF1); the human protein sequence correspondsto reference: Q81WU6 or Extracellular sulfatase Sulf-1 precursor(HSulf-1) gi|27356932|gb|AAM76860.1| extracellular sulfatase SULF-1[Homo sapiens].

The disrupted mouse gene is Sulf1 (sulfatase 1), ortholog of humanSULF1. Aliases include AW121680, extracellular sulfatase SULF-1, SULF-1,HSULF-1, KIAA1077, and sulfatase FP.

SULF1 is a secreted arylsulfatase with the ability to remove sulfatefrom glucosamine within subregions of heparin. This action on heparin islikely to inhibit signaling by heparin-dependent growth factors, therebyinhibiting cell proliferation and facilitating apoptosis(Morimoto-Tomita, et al., J Biol Chem, 277(51):49175-85 (2002); Lai etal., J Biol Chem, 278(25):23107-17 (2003); Lai et al., Gastroenterology,126(1):231-48 (2004)). Down-regulation of SULF1 may be a mechanism bywhich certain types of cancer cells enhance growth factor signaling (Laiet al., J Biol Chem, 278(25):23107-17 (2003)).

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 31 44 19 94 Expected 23.5 47 23.5 94 Chi-Sq. =3.45 Significance = 0.17846 (hom/n) = 0.20 Avg. Litter Size = 9

Mutation Type: Retroviral Insertion (OST)

Retroviral insertion occurred in the intron preceding coding exon 1(NCBI accession NM_(—)172294.1).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-208).

35.32.1. Phenotypic Analysis (for Disrupted Gene: DNA347767 (UNQ14964)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human sulfatase 1 (SULF1)resulted in a decreased IL-6, TNF alpha and MCP-1 response to LPSchallenge in (−/−) mice. Transcript was absent by RT-PCR.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACSCalibur instrument.

Results:

The (−/−) mice exhibited a decreased mean serum IL-6 response (as wellas TNF-alpha and MCP-1) to LPS challenge when compared with their (+/+)littermates. Analyzed wt/het/hom: 8/4/8

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO61709 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited a decreasedability to elicit an immunological response (TNF-alpha, MCP-1 and IL-6production) when challenged with the LPS endotoxin indicating aproinflammatory response. IL-6, MCP-1 and TNF alpha contribute to thelater stages of B cell activation. In addition, they play a criticalrole in inducing the acute phase response and systemic inflammation.This suggests that PRO61709 polypeptides or agonists thereof wouldstimulate the immune system and would find utility in the cases whereinthis effect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immunocompromised patients,such as AIDS sufferers. Accordingly, inhibitors or antagonists toPRO61709 polypeptides thereof would be useful in inhibiting the immuneresponse and would be useful candidates for suppressing harmful immuneresponses, e.g. in the case of graft rejection or graft-versus-hostdiseases.

35.33. Generation and Analysis of Mice Comprising DNA58801-1052 (UNQ455)Gene Disruptions

In these knockout experiments, the gene encoding PRO779 polypeptides(designated as DNA58801-1052 (UNQ455) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)033042 or ACCESSION:NM_(—)033042 NID: 14719437 Mus musculus Mus musculus tumor necrosisfactor receptor superfamily, member 12 (Tnfrsf12); protein reference:Q99MM1 or Q99MM1 Q99MM1 WSL-1-LIKE PROTEIN; the human gene sequencereference: NM_(—)003790 or ACCESSION: NM_(—)003790 NID: 4507568 Homosapiens Homo sapiens tumor necrosis factor receptor superfamily, member12 (translocating chain-association membrane protein) (TNFRSF12); thehuman protein sequence corresponds to reference: Q93038 or TR12_HUMANQ93038 WSL-1 PROTEIN PRECURSOR APOPTOSIS-MEDI.

The mouse gene of interest is Tnfrsf25 (tumor necrosis factor receptorsuperfamily, member 25), ortholog of human TNFRSF25. Aliases includeDR3, TR3, Wsl, DDR3, LARD, APO-3, TRAMP, WSL-1, WSL-LR, Tnfrsf12, WSL-1protein, death receptor beta, death domain receptor 3, apoptosisinducing receptor, apoptosis-mediating receptor, death domain receptor 3soluble form, lymphocyte associated receptor of death, translocatingchain-association membrane protein, and tumor necrosis factor receptorsuperfamily member 12. TNFRSF25 is a type I plasma membrane proteinexpressed in thymocytes and lymphocytes that functions as a receptor forthe ligands TNFSF12 (tumor necrosis factor (ligand) superfamily, member12) and TNFSF15 (tumor necrosis factor (ligand) superfamily, member 15).Activation of TNFRSF25 can stimulate apoptosis or, conversely, promote Tcell expansion by a nuclear factor kappa-B-mediated process (Chinnaiyanet al, Science 274(5289):990-2 (1996); Kitson et al, Nature384(6607):372-5 (1996); Migone et al, Immunity 16(3):479-92 (2002); Wenet al, J Biol Chem 278(40):39251-8 (2003)). TNFRSF25 appears to play arole in lymphocyte homeostasis. Wang et al., Mol Cell Biol21(10):3451-61 (2001) showed that negative selection andanti-CD3-induced apoptosis are impaired in TNFRSF25 homozygous null micebut not in wild-type mice, suggesting that TNFRSF25 plays a nonredundant role in removal of self-reactive T cells in the thymus.

Targeted or gene trap mutations were generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice werebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny were intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice were obtained from the chimera, F1 heterozygous mice werecrossed to 129SvEv^(Brd)/C57 hybrid mice to yield additionalheterozygous animals for the intercross to generate the F2 mice.

Level I phenotypic analysis is performed on mice from this generation

wt het hom Total Observed 21 42 21 84 Expected 21 42 21 84 Chi-Sq. =0.00 Significance = 1.00000 (hom/n) = 0.25 Avg. Litter Size = 8Mutation Type: Homologous Recombination (standard)Coding exons 1 through 5 were targeted (NCBI accession NM_(—)033042.2).Wild-type expression of the target gene was detected in embryonic stem(ES) cells and in all 13 adult tissue samples tested by RT-PCR.RT-PCR analysis revealed that the transcript was absent in the (−/−)mouse analyzed (M-208).

35.33.1. Phenotypic Analysis (for Disrupted Gene: DNA58801-1052 (UNQ455)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human tumor necrosisfactor receptor superfamily, member 25 (TNFRSF25) resulted in decreasedlumbar 5 vertebra measurements in the (−/−) mice compared to thewild-type littermate controls and historical means. In addition, themutant knockout mice exhibited an enhanced IgG responses to an ovalbuminchallenge.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmunodominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Analyzed wt/het/hom: 8/4/9

Results of this Challenge:

The (−/−) mice exhibited an increased mean serum IgG2a response toovalbumin challenge when compared with their (+/+) littermates and thehistorical means. Thus, these knockout mice exhibited an increasedability to elicit an OVA-specific antibody response to the T-celldependent OVA antigen.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO779 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited an increased ability to elicit animmunological response when challenged with the T-cell dependent OVAantigen. Thus, inhibitors or antagonists of PRO779 polypeptides would beuseful for stimulating the immune system (such as T cell proliferation)and would find utility in the cases wherein this effect would bebeneficial to the individual such as in the case of leukemia, and othertypes of cancer, and in immunocompromised patients, such as AIDSsufferers. Accordingly, PRO779 polypeptides or agonists thereof would beuseful for inhibiting the immune response and thus would be usefulcandidates for suppressing harmful immune responses, e.g. in the case ofgraft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygotes and 8 homozygoteswere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro-CT: The (−/−) mice exhibited a decreased lumbar 5 vertebrameasurements in bone volume, trabecular number and connectivity densitywhen compared with their gender-matched (+/+) littermates and thehistorical means. These results demonstrate that knockout mutant miceexhibit abnormal bone metabolism with significant bone loss similar toosteoporosis characterized by decrease in bone mass with decreaseddensity and possibly fragility leading to bone fractures. Thus, itappears that PRO779 polypeptides or agonists thereof play a role inmaintaining bone homeostasis. In addition, PRO779 or its encoding genewould be useful in maintaining bone homeostasis and would be importantin bone healing or useful for the treatment of osteoarthritis orosteoporosis; whereas antagonists to PRO779 or its encoding gene wouldlead to abnormal or pathological bone disorders including inflammatorydiseases associated with abnormal bone metabolism including arthritis,osteoporosis, and osteopenia.

Example 36 Use of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 as a hybridization probe

The following method describes use of a nucleotide sequence encoding aPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide as a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptides as disclosed herein is employed as a probe to screen forhomologous DNAs (such as those encoding naturally-occurring variants ofPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides) in human tissue cDNA libraries or human tissuegenomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-,PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-,PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-,PRO7425-, PRO10102-, PRO10282-, PRO61709- or PRO779-derived probe to thefilters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS,0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt'ssolution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of thefilters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides can then be identified usingstandard techniques known in the art.

Example 37 Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides by recombinant expression in E. coli.

The DNA sequence encoding a PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 coding region, lambdatranscriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide are pooled and theacetonitrile removed using a gentle stream of nitrogen directed at thesolution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 Msodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Example 38 Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 in mammalian cells

This example illustrates preparation of a potentially glycosylated formof a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide by recombinant expression in mammaliancells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO196, pRK5-PRO217, pRK5-PRO231,pRK5-PRO236, pRK5-PRO245, pRK5-PRO246, pRK5-PRO258, pRK5-PRO287,pRK5-PRO328, pRK5-PRO344, pRK5-PRO357, pRK5-PRO526, pRK5-PRO724,pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104, pRK5-PRO1151,pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570, pRK5-PRO1886,pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994, pRK5-PRO6097,pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282, pRK5-PRO61709 orpRK5-PRO779.

The selected host cells may be 293 cells. Human 293 cells (ATCC CCL1573) are grown to confluence in tissue culture plates in medium such asDMEM supplemented with fetal calf serum and optionally, nutrientcomponents and/or antibiotics. About 10 μg pRK5-PRO196, pRK5-PRO217,pRK5-PRO231, pRK5-PRO236, pRK5-PRO245, pRK5-PRO246, pRK5-PRO258,pRK5-PRO287, pRK5-PRO328, pRK5-PRO344, pRK5-PRO357, pRK5-PRO526,pRK5-PRO724, pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104,pRK5-PRO1151, pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570,pRK5-PRO1886, pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994,pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282, pRK5-PRO61709or pRK5-PRO779 DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptides. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO196, pRK5-PRO217,pRK5-PRO231, pRK5-PRO236, pRK5-PRO245, pRK5-PRO246, pRK5-PRO258,pRK5-PRO287, pRK5-PRO328, pRK5-PRO344, pRK5-PRO357, pRK5-PRO526,pRK5-PRO724, pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104,pRK5-PRO1151, pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570,pRK5-PRO1886, pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994,pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282, pRK5-PRO61709or pRK5-PRO779 DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 canthen be concentrated and purified by any selected method, such asdialysis and/or column chromatography.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 can be expressed in CHO cells. The pRK5-PRO196, pRK5-PRO217,pRK5-PRO231, pRK5-PRO236, pRK5-PRO245, pRK5-PRO246, pRK5-PRO258,pRK5-PRO287, pRK5-PRO328, pRK5-PRO344, pRK5-PRO357, pRK5-PRO526,pRK5-PRO724, pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104,pRK5-PRO1151, pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570,pRK5-PRO1886, pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994,pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282, pRK5-PRO61709or pRK5-PRO779 can be transfected into CHO cells using known reagentssuch as CaPO₄ or DEAE-dextran. As described above, the cell cultures canbe incubated, and the medium replaced with culture medium (alone) ormedium containing a radiolabel such as ³⁵S-methionine. After determiningthe presence of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 can then be concentrated and purified byany selected method.

Epitope-tagged PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 may also be expressed in host CHO cells. The PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 may besubcloned out of the pRK5 vector. The subclone insert can undergo PCR tofuse in frame with a selected epitope tag such as a poly-his tag into aBaculovirus expression vector. The poly-his tagged PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determinedOn day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 39 Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 in Yeast

The following method describes recombinant expression of PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 from the ADH2/GAPDH promoter. DNA encodingPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression ofPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779. For secretion, DNA encoding PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 may further be purified using selectedcolumn chromatography resins.

Example 40 Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 inBaculovirus-infected insect cells.

The sequence coding for PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 or the desired portion of the codingsequence of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 such as the sequence encoding the extracellulardomain of a transmembrane protein or the sequence encoding the matureprotein if the protein is extracellular is amplified by PCR with primerscomplementary to the 5′ and 3′ regions. The 5′ primer may incorporateflanking (selected) restriction enzyme sites. The product is thendigested with those selected restriction enzymes and subcloned into theexpression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Example 41 Tissue Expression Profiling Using GeneExpress®

A proprietary database containing gene expression information(GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in anattempt to identify polypeptides (and their encoding nucleic acids)whose expression is significantly upregulated in a particular tumortissue(s) of interest as compared to other tumor(s) and/or normaltissues. Specifically, analysis of the GeneExpress® database wasconducted using either software available through Gene Logic Inc.,Gaithersburg, Md., for use with the GeneExpress® database or withproprietary software written and developed at Genentech, Inc. for usewith the GeneExpress® database. The rating of positive hits in theanalysis is based upon several criteria including, for example, tissuespecificity, tumor specificity and expression level in normal essentialand/or normal proliferating tissues. The following is a list ofmolecules whose tissue expression profile as determined from an analysisof the GeneExpress® database evidences high tissue expression andsignificant upregulation of expression in a specific tumor or tumors ascompared to other tumor(s) and/or normal tissues and optionallyrelatively low expression in normal essential and/or normalproliferating tissues. Tissue expression profiling was performed onseveral UNQ genes the results of which are disclosed in Example 35.

Example 42 Microarray Analysis to Detect Upregulation of UNQ Genes inCancerous Tumors

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in diseasedtissues as compared to their normal counterparts. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes known to be expressed in certain disease states may be arrayedon a solid support. Hybridization of a labeled probe with a particulararray member indicates that the sample from which the probe was derivedexpresses that gene. If the hybridization signal of a probe from a test(disease tissue) sample is greater than hybridization signal of a probefrom a control (normal tissue) sample, the gene or genes overexpressedin the disease tissue are identified. The implication of this result isthat an overexpressed protein in a diseased tissue is useful not only asa diagnostic marker for the presence of the disease condition, but alsoas a therapeutic target for treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In one example, the specificpreparation of nucleic acids for hybridization and probes, slides, andhybridization conditions are all detailed in PCT Patent ApplicationSerial No. PCT/US01/10482, filed on Mar. 30, 2001 and which is hereinincorporated by reference.

In the present example, cancerous tumors derived from various humantissues were studied for upregulated gene expression relative tocancerous tumors from different tissue types and/or non-cancerous humantissues in an attempt to identify those polypeptides which areoverexpressed in a particular cancerous tumor(s). In certainexperiments, cancerous human tumor tissue and non-cancerous human tumortissue of the same tissue type (often from the same patient) wereobtained and analyzed for UNQ polypeptide expression. Additionally,cancerous human tumor tissue from any of a variety of different humantumors was obtained and compared to a “universal” epithelial controlsample which was prepared by pooling non-cancerous human tissues ofepithelial origin, including liver, kidney, and lung. mRNA isolated fromthe pooled tissues represents a mixture of expressed gene products fromthese different tissues. Microarray hybridization experiments using thepooled control samples generated a linear plot in a 2-color analysis.The slope of the line generated in a 2-color analysis was then used tonormalize the ratios of (test:control detection) within each experiment.The normalized ratios from various experiments were then compared andused to identify clustering of gene expression. Thus, the pooled“universal control” sample not only allowed effective relative geneexpression determinations in a simple 2-sample comparison, it alsoallowed multi-sample comparisons across several experiments.

In the present experiments, nucleic acid probes derived from the hereindescribed UNQ polypeptide-encoding nucleic acid sequences were used inthe creation of the microarray and RNA from various tumor tissues wereused for the hybridization thereto. Below is shown the results of theseexperiments, demonstrating that various UNQ polypeptides of the presentinvention are significantly overexpressed in various human tumor tissuesas compared to their normal counterpart tissue(s). Moreover, all of themolecules shown below are significantly overexpressed in their specifictumor tissue(s) as compared to in the “universal” epithelial control. Asdescribed above, these data demonstrate that the UNQ polypeptides of thepresent invention are useful not only as diagnostic markers for thepresence of one or more cancerous tumors, but also serve as therapeutictargets for the treatment of those tumors. Microarray analysis wasperformed on several UNQ genes the results of which are disclosed inExample 35.

Example 43 Quantitative Analysis of UNQ mRNA Expression

In this assay, a 5′ nuclease assay (for example, TaqMan0) and real-timequantitative PCR (for example, ABI Prizm 7700 Sequence Detection System®(Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), wereused to find genes that are significantly overexpressed in a canceroustumor or tumors as compared to other cancerous tumors or normalnon-cancerous tissue. The 5′ nuclease assay reaction is a fluorescentPCR-based technique which makes use of the 5′ exonuclease activity ofTaq DNA polymerase enzyme to monitor gene expression in real time. Twooligonucleotide primers (whose sequences are based upon the gene or ESTsequence of interest) are used to generate an amplicon typical of a PCRreaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the PCR amplification reaction, the Taq DNApolymerase enzyme cleaves the probe in a template-dependent manner. Theresultant probe fragments disassociate in solution, and signal from thereleased reporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700™ Sequence Detection. The system consists of athermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

The starting material for the screen was mRNA isolated from a variety ofdifferent cancerous tissues. The mRNA is quantitated precisely, e.g.,fluorometrically. As a negative control, RNA was isolated from variousnormal tissues of the same tissue type as the cancerous tissues beingtested.

5′ nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ACt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer mRNA results to normal human mRNA results. As one Ctunit corresponds to 1 PCR cycle or approximately a 2-fold relativeincrease relative to normal, two units corresponds to a 4-fold relativeincrease, 3 units corresponds to an 8-fold relative increase and so on,one can quantitatively measure the relative fold increase in mRNAexpression between two or more different tissues. Using this technique,the molecules have been identified as being significantly overexpressedin a particular tumor(s) as compared to their normal non-cancerouscounterpart tissue(s) (from both the same and different tissue donors)and thus, represent excellent polypeptide targets for the diagnosis andtherapy of cancer in mammals. Specific results for a UNQ gene aredisclosed in Example 35.

Example 44 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10μ; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the probe was immediately placed on ice.The wells of gel were flushed, the sample loaded, and run at 180-250volts for 45 minutes. The gel was wrapped in saran wrap and exposed toXAR film with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)-human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)-saturated filter paper.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, Vf=4 L), followed by RNaseA treatment at 37°C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20 μg/ml),The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, Vf=4 L).

F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses were obtainedso as to be complementary to the nucleic acids (or the complementsthereof) as shown in the accompanying figures.

G. Results

In situ analysis was performed on a variety of DNA sequences disclosedherein the results of which are disclosed in Example 35.

Example 45 Preparation of Antibodies that Bind PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides, fusion proteins containingPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides, and cells expressing recombinant PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides onthe cell surface. Selection of the immunogen can be made by the skilledartisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779. Three to four days later, the mice are sacrificedand the spleen cells are harvested. The spleen cells are then fused(using 35% polyethylene glycol) to a selected murine myeloma cell linesuch as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO196,anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,anti-PRO61709 or anti-PRO779 monoclonal antibodies. Alternatively, thehybridoma cells can be grown in tissue culture flasks or roller bottles.Purification of the monoclonal antibodies produced in the ascites can beaccomplished using ammonium sulfate precipitation, followed by gelexclusion chromatography. Alternatively, affinity chromatography basedupon binding of antibody to protein A or protein G can be employed.

Example 46 Purification of PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 Polypeptides Using Specific Antibodies

Native or recombinant PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptides may be purified by a varietyof standard techniques in the art of protein purification. For example,pro-PRO196, pro-PRO217, pro-PRO231, pro-PRO236, pro-PRO245, pro-PRO246,pro-PRO258, pro-PRO287, pro-PRO328, pro-PRO344, pro-PRO357, pro-PRO526,pro-PRO724, pro-PRO731, pro-PRO732, pro-PRO1003, pro-PRO1104,pro-PRO1151, pro-PRO1244, pro-PRO1298, pro-PRO1313, pro-PRO1570,pro-PRO1886, pro-PRO1891, pro-PRO4409, pro-PRO5725, pro-PRO5994,pro-PRO6097, pro-PRO7425, pro-PRO10102, pro-PRO10282, pro-PRO61709 orpro-PRO779 polypeptide, mature PRO196, mature PRO217, mature PRO231,mature PRO236, mature PRO245, mature PRO246, mature PRO258, maturePRO287, mature PRO328, mature PRO344, mature PRO357, mature PRO526,mature PRO724, mature PRO731, mature PRO732, mature PRO1003, maturePRO1104, mature PRO1151, mature PRO1244, mature PRO1298, mature PRO1313,mature PRO1570, mature PRO1886, mature PRO1891, mature PRO4409, maturePRO5725, mature PRO5994, mature PRO6097, mature PRO7425, maturePRO10102, mature PRO10282, mature PRO61709 or mature PRO779 polypeptide,or pre-PRO196, pre-PRO217, pre-PRO231, pre-PRO236, pre-PRO245,pre-PRO246, pre-PRO258, pre-PRO287, pre-PRO328, pre-PRO344, pre-PRO357,pre-PRO526, pre-PRO724, pre-PRO731, pre-PRO732, pre-PRO1003,pre-PRO1104, pre-PRO1151, pre-PRO1244, pre-PRO1298, pre-PRO1313,pre-PRO1570, pre-PRO1886, pre-PRO1891, pre-PRO4409, pre-PRO5725,pre-PRO5994, pre-PRO6097, pre-PRO7425, pre-PRO10102, pre-PRO10282,pre-PRO61709 or pre-PRO779 polypeptide is purified by immunoaffinitychromatography using antibodies specific for the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide of interest.In general, an immunoaffinity column is constructed by covalentlycoupling the anti-PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide antibody to an activatedchromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide by preparing a fraction from cells containing PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO196, antibody/PRO217,antibody/PRO231, antibody/PRO236, antibody/PRO245, antibody/PRO246,antibody/PRO258, antibody/PRO287, antibody/PRO328, antibody/PRO344,antibody/PRO357, antibody/PRO526, antibody/PRO724, antibody/PRO731,antibody/PRO732, antibody/PRO1003, antibody/PRO1104, antibody/PRO1151,antibody/PRO1244, antibody/PRO1298, antibody/PRO1313, antibody/PRO1570,antibody/PRO1886, antibody/PRO1891, antibody/PRO4409, antibody/PRO5725,antibody/PRO5994, antibody/PRO6097, antibody/PRO7425, antibody/PRO10102,antibody/PRO10282, antibody/PRO61709 or antibody/PRO779 polypeptidebinding (e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), andPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide is collected.

Example 47 Drug Screening

This invention is particularly useful for screening compounds by usingPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptides or binding fragment thereof in any of a variety ofdrug screening techniques. The PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide-associated disease ordisorder. These methods comprise contacting such an agent with anPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide or fragment thereof and assaying (I) for the presenceof a complex between the agent and the PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or fragment, or (ii)for the presence of a complex between the PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide or fragment is typicallylabeled. After suitable incubation, free PRO196, PRO217, PRO231, PRO236,PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or fragment isseparated from that present in bound form, and the amount of free oruncomplexed label is a measure of the ability of the particular agent tobind to PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide or to interfere with the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide, the peptide test compounds arereacted with PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide and washed. Bound PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide is detectedby methods well known in the art. Purified PRO196, PRO217, PRO231,PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. In addition, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide specifically compete with a test compound for binding toPRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide or fragments thereof. In this manner, the antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants with PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide.

Example 48 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide orwhich enhance or interfere with the function of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, orof a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,PRO61709 or PRO779 polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thePRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 orPRO779 polypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO196, PRO217, PRO231, PRO236, PRO245,PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,PRO10282, PRO61709 or PRO779 polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide-like molecules or to identify efficient inhibitors. Usefulexamples of rational drug design may include molecules which haveimproved activity or stability as shown by Braxton and Wells,Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists,or antagonists of native peptides as shown by Athauda et al., J.Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO196,PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779polypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO196, PRO217,PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

1. A method of identifying a phenotype associated with a disruption of agene which encodes for a PRO258 polypeptide, the method comprising: (a)providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO258 polypeptide; (b)measuring a physiological characteristic of the non-human transgenicanimal; and (c) comparing the measured physiological characteristic withthat of a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal.
 2. The method of claim 1, wherein the non-human transgenicanimal is heterozygous for the disruption of a gene which encodes for aPRO258 polypeptide.
 3. The method of claim 1, wherein the phenotypeexhibited by the non-human transgenic animal as compared with gendermatched wild-type littermates is at least one of the following: aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an immunological disorder; a bone metabolic abnormality ordisorder; or a lipid metabolic disorder. 4-5. (canceled)
 6. The methodof claim 3, wherein the neurological disorder is an abnormal circadianrhythm during home-cage activity testing.
 7. The method of claim 3,wherein the neurological disorder is an enhanced motor coordinationduring inverted screen testing. 8-17. (canceled)
 18. The method of claim3, wherein the cardiovascular, endothelial or angiogenic disorders arearterial diseases, such as diabetes mellitus; papilledema; opticatrophy; atherosclerosis; angina; myocardial infarctions such as acutemyocardial infarctions, cardiac hypertrophy, and heart failure such ascongestive heart failure; hypertension; inflammatory vasculitides;Reynaud's disease and Reynaud's phenomenon; aneurysms and arterialrestenosis; venous and lymphatic disorders such as thrombophlebitis,lymphangitis, and lymphedema; peripheral vascular disease; cancer suchas vascular tumors, e.g., hemangioma (capillary and cavernous), glomustumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma; tumor angiogenesis; trauma such as wounds, burns, andother injured tissue, implant fixation, scarring; ischemia reperfusioninjury; rheumatoid arthritis; cerebrovascular disease; renal diseasessuch as acute renal failure, or osteoporosis.
 19. The method of claim 3,wherein the immunological disorders are systemic lupus erythematosis;rheumatoid arthritis; juvenile chronic arthritis; spondyloarthropathies;systemic sclerosis (scleroderma); idiopathic inflammatory myopathies(dermatomyositis, polymyositis); Sjögren's syndrome; systemicvasculitis; sarcoidosis; autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory bowel disease (ulcerative colitis: Crohn's disease);gluten-sensitive enteropathy, and Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; or transplantation associated diseases including graftrejection and graft-versus-host disease.
 20. The method of claim 3,wherein the bone metabolic abnormality or disorder is arthritis,osteoporosis or osteopetrosis.
 21. The method of claim 1, wherein thenon-human transgenic animal exhibits at least one of the followingphysiological characteristics compared with gender matched wild-typelittermates: abnormal circadian rhythm during home-cage activity testingincluding decreased ambulatory counts; enhanced motor coordinationduring inverted screen testing; increased mean fasting serum glucoselevels; decreased heart rate; increased mean serum cholesterol levels;increased mean percentage of CD4 cells and decreased mean percentage ofB cells; decreased mean serum IgG2a response to an ovalbumin challenge;decreased mean total white blood cell (WBC) counts; decreased absolutelymphocyte counts; decreased absolute monocyte counts; decreased totalfemoral midshaft cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density.
 22. An isolatedcell derived from a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO258 polypeptide.
 23. Theisolated cell of claim 22 which is a murine cell.
 24. The isolated cellof claim 23, wherein the murine cell is an embryonic stem cell.
 25. Theisolated cell of claim 22, wherein the non-human transgenic animalexhibits at least one of the following phenotypes compared with gendermatched wild-type littermates: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an immunologicaldisorder; a bone metabolic abnormality or disorder; or a lipid metabolicdisorder.
 26. A method of identifying an agent that modulates aphenotype associated with a disruption of a gene which encodes for aPRO258 polypeptide, the method comprising: (a) providing a non-humantransgenic animal whose genome comprises a disruption of the gene whichencodes for the PRO258 polypeptide; (b) measuring a physiologicalcharacteristic of the non-human transgenic animal of (a); (c) comparingthe measured physiological characteristic of (b) with that of a gendermatched wild-type animal, wherein the physiological characteristic ofthe non-human transgenic animal that differs from the physiologicalcharacteristic of the wild-type animal is identified as a phenotyperesulting from the gene disruption in the non-human transgenic animal;(d) administering a test agent to the non-human transgenic animal of(a); and (e) determining whether the test agent modulates the identifiedphenotype associated with gene disruption in the non-human transgenicanimal.
 27. The method of claim 26, wherein the phenotype associatedwith the gene disruption comprises a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality. 28-29. (canceled)
 30. The method of claim 27, wherein theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing.
 31. The method of claim 27, wherein the neurologicaldisorder is an enhanced motor coordination during inverted screentesting. 32-41. (canceled)
 42. The method of claim 27, wherein thecardiovascular, endothelial or angiogenic disorders are arterialdiseases, such as diabetes mellitus; papilledema; optic atrophy;atherosclerosis; angina; myocardial infarctions such as acute myocardialinfarctions, cardiac hypertrophy, and heart failure such as congestiveheart failure; hypertension; inflammatory vasculitides; Reynaud'sdisease and Reynaud's phenomenon; aneurysms and arterial restenosis;venous and lymphatic disorders such as thrombophlebitis, lymphangitis,and lymphedema; peripheral vascular disease; cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma; tumor angiogenesis; trauma such as wounds, burns, andother injured tissue, implant fixation, scarring; ischemia reperfusioninjury; rheumatoid arthritis; cerebrovascular disease; renal diseasessuch as acute renal failure, or osteoporosis.
 43. The method of claim27, wherein the immunological disorders are systemic lupuserythematosis; rheumatoid arthritis; juvenile chronic arthritis;spondyloarthropathies; systemic sclerosis (scleroderma); idiopathicinflammatory myopathies (dermatomyositis, polymyositis); Sjögren'ssyndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory bowel disease (ulcerative colitis: Crohn's disease);gluten-sensitive enteropathy, and Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; or transplantation-associated diseases including graftrejection and graft-versus-host disease.
 44. The method of claim 27,wherein said bone metabolic abnormality or disorder is arthritis,osteoporosis or osteopetrosis.
 45. The method of claim 26, wherein thenon-human transgenic animal exhibits at least one of the followingphysiological characteristics compared with gender matched wild-typelittermates: abnormal circadian rhythm during home-cage activity testingincluding decreased ambulatory counts; enhanced motor coordinationduring inverted screen testing; increased mean fasting serum glucoselevels; decreased heart rate; increased mean serum cholesterol levels;increased mean percentage of CD4 cells and decreased mean percentage ofB cells; decreased mean serum IgG2a response to an ovalbumin challenge;decreased mean total white blood cell (WBC) counts; decreased absolutelymphocyte counts; decreased absolute monocyte counts; decreased totalfemoral midshaft cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density. 46-49.(canceled)
 50. A method of identifying an agent that modulates aphysiological characteristic associated with a disruption of the genewhich encodes for a PRO258 polypeptide, the method comprising: (a)providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO258 polypeptide; (b)measuring a physiological characteristic exhibited by the non-humantransgenic animal of (a); (c) comparing the measured physiologicalcharacteristic of (b) with that of a gender matched wild-type animal,wherein the physiological characteristic exhibited by the non-humantransgenic animal that differs from the physiological characteristicexhibited by the wild-type animal is identified as a physiologicalcharacteristic associated with gene disruption; (d) administering a testagent to the non-human transgenic animal of (a); and (e) determiningwhether the physiological characteristic associated with gene disruptionis modulated.
 51. The method of claim 50, wherein the non-humantransgenic animal exhibits at least one of the following physiologicalcharacteristics compared with gender matched wild-type littermates:abnormal circadian rhythm during home-cage activity testing includingdecreased ambulatory counts; enhanced motor coordination during invertedscreen testing; increased mean fasting serum glucose levels; decreasedheart rate; increased mean serum cholesterol levels; increased meanpercentage of CD4 cells and decreased mean percentage of B cells;decreased mean serum IgG2a response to an ovalbumin challenge; decreasedmean total white blood cell (WBC) counts; decreased absolute lymphocytecounts; decreased absolute monocyte counts; decreased total femoralmidshaft cross-sectional area; decreased mean vertebral trabecular bonevolume, number and connectivity density. 52-55. (canceled)
 56. A methodof identifying an agent which modulates a behavior associated with adisruption of the gene which encodes for a PRO258 polypeptide, themethod comprising: (a) providing a non-human transgenic animal whosegenome comprises a disruption of the gene which encodes for a PRO258polypeptide; (b) observing the behavior exhibited by the non-humantransgenic animal of (a); (c) comparing the observed behavior of (b)with that of a gender matched wild-type animal, wherein the observedbehavior exhibited by the non-human transgenic animal that differs fromthe observed behavior exhibited by the wild-type animal is identified asa behavior associated with gene disruption; (d) administering a testagent to the non-human transgenic animal of (a); and (e) determiningwhether the agent modulates the behavior associated with genedisruption. 57-58. (canceled)
 59. The method of claim 56, wherein thebehavior is an abnormal circadian rhythm during home-cage activitytesting.
 60. The method of claim 56, wherein the behavior is an enhancedmotor coordination during inverted screen testing. 61-66. (canceled) 67.A method of identifying an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an immunological disorder; a bone metabolic abnormality ordisorder; or a lipid metabolic disorder associated with a disruption inthe gene which encodes for a PRO258 polypeptide, the method comprising:(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO258 polypeptide; (b)administering a test agent to said non-human transgenic animal; and (c)determining whether said test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; immunological disorder; bone metabolic abnormality ordisorder; or lipid metabolic disorder; in the non-human transgenicanimal. 68-69. (canceled)
 70. The method of claim 67, wherein theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing.
 71. The method of claim 67, wherein the neurologicaldisorder is an enhanced motor coordination during inverted screentesting. 72-81. (canceled)
 82. The method of claim 67, wherein thecardiovascular, endothelial or angiogenic disorders are arterialdiseases, such as diabetes mellitus; papilledema; optic atrophy;atherosclerosis; angina; myocardial infarctions such as acute myocardialinfarctions, cardiac hypertrophy, and heart failure such as congestiveheart failure; hypertension; inflammatory vasculitides; Reynaud'sdisease and Reynaud's phenomenon; aneurysms and arterial restenosis;venous and lymphatic disorders such as thrombophlebitis, lymphangitis,and lymphedema; peripheral vascular disease; cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma; tumor angiogenesis; trauma such as wounds, burns, andother injured tissue, implant fixation, scarring; ischemia reperfusioninjury; rheumatoid arthritis; cerebrovascular disease; renal diseasessuch as acute renal failure, or osteoporosis.
 83. The method of claim67, wherein the immunological disorders are systemic lupuserythematosis; rheumatoid arthritis; juvenile chronic arthritis;spondyloarthropathies; systemic sclerosis (scleroderma); idiopathicinflammatory myopathies (dermatomyositis, polymyositis); Sjögren'ssyndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory bowel disease (ulcerative colitis: Crohn's disease);gluten-sensitive enteropathy, and Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; or transplantation associated diseases including graftrejection and graft-versus-host disease.
 84. The method of claim 67,wherein said bone metabolic abnormality or disorder is arthritis,osteoporosis or osteopetrosis.
 85. The method of claim 67, wherein thenon-human transgenic animal exhibits at least one of the followingphysiological characteristics compared with gender matched wild-typelittermates: abnormal circadian rhythm during home-cage activity testingincluding decreased ambulatory counts; enhanced motor coordinationduring inverted screen testing; increased mean fasting serum glucoselevels; decreased heart rate; increased mean serum cholesterol levels;increased mean percentage of CD4 cells and decreased mean percentage ofB cells; decreased mean serum IgG2a response to an ovalbumin challenge;decreased mean total white blood cell (WBC) counts; decreased absolutelymphocyte counts; decreased absolute monocyte counts; decreased totalfemoral midshaft cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density. 86-90.(canceled)
 91. A method of identifying an agent that modulates theexpression of a PRO258 polypeptide, the method comprising: (a)contacting a test agent with a host cell expressing a PRO258polypeptide; and (b) determining whether the test agent modulates theexpression of the PRO258 polypeptide by the host cell. 92-95. (canceled)96. A method of evaluating a therapeutic agent capable of affecting acondition associated with a disruption of a gene which encodes for aPRO258 polypeptide, the method comprising: (a) providing a non-humantransgenic animal whose genome comprises a disruption of the gene whichencodes for the PRO258 polypeptide; (b) measuring a physiologicalcharacteristic of the non-human transgenic animal of (a); (c) comparingthe measured physiological characteristic of (b) with that of a gendermatched wild-type animal, wherein the physiological characteristic ofthe non-human transgenic animal that differs from the physiologicalcharacteristic of the wild-type animal is identified as a conditionresulting from the gene disruption in the non-human transgenic animal;(d) administering a test agent to the non-human transgenic animal of(a); and (e) evaluating the effects of the test agent on the identifiedcondition associated with gene disruption in the non-human transgenicanimal.
 97. The method of claim 96, wherein the condition is aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an immunological disorder; a bone metabolic abnormality ordisorder; or a lipid metabolic disorder. 98-102. (canceled)
 103. Amethod of treating or preventing or ameliorating a neurologicaldisorder; cardiovascular, endothelial or angiogenic disorder;immunological disorder; bone metabolic abnormality or disorder; or lipidmetabolic disorder associated with the disruption of a gene whichencodes for a PRO258 polypeptide, the method comprising administering toa subject in need of such treatment whom may already have the disorder,or may be prone to have the disorder or may be in whom the disorder isto be prevented, a therapeutically effective amount of therapeutic agentidentified by the method of claim 91, or agonists or antagoniststhereof, thereby effectively treating or preventing or ameliorating saiddisorder. 104-105. (canceled)
 106. The method of claim 103, wherein theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing.
 107. The method of claim 103, wherein the neurologicaldisorder is an enhanced motor coordination during inverted screentesting. 108-117. (canceled)
 118. The method of claim 103, wherein thecardiovascular, endothelial or angiogenic disorders are arterialdiseases, such as diabetes mellitus; papilledema; optic atrophy;atherosclerosis; angina; myocardial infarctions such as acute myocardialinfarctions, cardiac hypertrophy, and heart failure such as congestiveheart failure; hypertension; inflammatory vasculitides; Reynaud'sdisease and Reynaud's phenomenon; aneurysms and arterial restenosis;venous and lymphatic disorders such as thrombophlebitis, lymphangitis,and lymphedema; peripheral vascular disease; cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma; tumor angiogenesis; trauma such as wounds, burns, andother injured tissue, implant fixation, scarring; ischemia reperfusioninjury; rheumatoid arthritis; cerebrovascular disease; renal diseasessuch as acute renal failure, or osteoporosis.
 119. The method of claim103, wherein the immunological disorders are systemic lupuserythematosis; rheumatoid arthritis; juvenile chronic arthritis;spondyloarthropathies; systemic sclerosis (scleroderma); idiopathicinflammatory myopathies (dermatomyositis, polymyositis); Sjögren'ssyndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory bowel disease (ulcerative colitis: Crohn's disease);gluten-sensitive enteropathy, and Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; or transplantation associated diseases including graftrejection and graft-versus-host disease.
 120. The method of claim 103,wherein said bone metabolic abnormality or disorder is arthritis,osteoporosis or osteopetrosis.
 121. A method of identifying an agentthat ameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an immunological disorder; a bonemetabolic abnormality or disorder; or a lipid metabolic disorderassociated with a disruption in the gene which encodes for a PRO258polypeptide, the method comprising: (a) providing a non-human transgenicanimal cell culture, each cell of said culture comprising a disruptionof the gene which encodes for a PRO258 polypeptide; (b) administering atest agent to said cell culture; and (c) determining whether said testagent ameliorates or modulates the neurological disorder;cardiovascular, endothelial or angiogenic disorder; eye abnormality;immunological disorder; oncological disorder; bone metabolic abnormalityor disorder; lipid metabolic disorder; or developmental abnormality insaid cell culture. 122-123. (canceled)
 124. The method of claim 121,wherein the neurological disorder is an abnormal circadian rhythm duringhome-cage activity testing.
 125. The method of claim 121, wherein theneurological disorder is an enhanced motor coordination during invertedscreen testing.
 126. (canceled)
 127. The method of claim 121, whereinthe neurological disorder is depression, generalized anxiety disorders,attention deficit disorder, sleep disorder, hyperactivity disorder,obsessive compulsive disorder, schizophrenia, cognitive disorders,hyperalgesia or sensory disorders. 128-135. (canceled)
 136. The methodof claim 121, wherein the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.
 137. The method of claim 121, wherein the immunologicaldisorders are systemic lupus erythematosis; rheumatoid arthritis;juvenile chronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.
 138. The methodof claim 121, wherein said bone metabolic abnormality or disorder isarthritis, osteoporosis or osteopetrosis. 139-143. (canceled)
 144. Amethod of modulating a phenotype associated with a disruption of a genewhich encodes for a PRO258 polypeptide, the method comprisingadministering to a subject whom may already have the phenotype, or maybe prone to have the phenotype or may be in whom the phenotype is to beprevented, an effective amount of an agent identified by the method ofclaim 26, or agonists or antagonists thereof, thereby effectivelymodulating the phenotype.
 145. A method of modulating a physiologicalcharacteristic associated with a disruption of a gene which encodes fora PRO258 polypeptide, the method comprising administering to a subjectwhom may already exhibit the physiological characteristic, or may beprone to exhibit the physiological characteristic or may be in whom thephysiological characteristic is to be prevented, an effective amount ofan agent identified by the method of claim 50, or agonists orantagonists thereof, thereby effectively modulating the physiologicalcharacteristic.
 146. A method of modulating a behavior associated with adisruption of a gene which encodes for a PRO258 polypeptide, the methodcomprising administering to a subject whom may already exhibit thebehavior, or may be prone to exhibit the behavior or may be in whom theexhibited behavior is to be prevented, an effective amount of an agentidentified by the method of claim 56, or agonists or antagoniststhereof, thereby effectively modulating the behavior.
 147. A method ofmodulating the expression of a PRO258 polypeptide, the method comprisingadministering to a host cell expressing said PRO258 polypeptide, aneffective amount of an agent identified by the method of claim 91, oragonists or antagonists thereof, thereby effectively modulating theexpression of said polypeptide.
 148. A method of modulating a conditionassociated with a disruption of a gene which encodes for a PRO258polypeptide, the method comprising administering to a subject whom mayhave the condition, or may be prone to have the condition or may be inwhom the condition is to be prevented, a therapeutically effectiveamount of a therapeutic agent identified by the method of claim 96, oragonists or antagonists thereof, thereby effectively modulating thecondition.
 149. A method of treating or preventing or ameliorating aneurological disorder; cardiovascular, endothelial or angiogenicdisorder; immunological disorder; bone metabolic abnormality ordisorder, or lipid metabloc disorder associated with the disruption of agene which encodes for a PRO258 polypeptide, the method comprisingadministering to a non-human transgenic animal cell culture, each cellof said culture comprising a disruption of the gene which encodes for aPRO258 polypeptide, a therapeutically effective amount of an agentidentified by the method of claim 121, or agonists or antagoniststhereof, thereby effectively treating or preventing or ameliorating saiddisorder.